Diagnosis and treatment of PTP04 related disorders

ABSTRACT

The present invention relates to PTP04 polypeptides, nucleic acids encoding such polypeptides, cells, tissues and animal containing such nucleic acids, antibodies to such polypeptides, assays utilizing such polypeptides, and methods relating to all of the foregoing. Methods for treatment, diagnosis, and screening are provided for PTP04 related diseases or conditions characterized by an abnormal interaction between a PTP04 binding partner.

RELATED APPLICATIONS

This application is a Divisional of application Ser. No. 09/081,345 fileon May 19, 1998, now U.S. Pat. No. 6,228,641 which claims priority tothe U.S. Provisional Patent Application No. 60/047,222, by Jallal etal., entitled “Diagnosis and Treatment of PTP04 elated Disorders.” andfiled May 20, 1997, which is incorporated herein by reference in itsentirety, including any drawings.

FIELD OF THE INVENTION

The present invention relates to tyrosine phosphatases. In particular,the invention concerns a protein we have named PTP04, nucleotidesequences encoding PTP04, various products and assay methods that can beused for identifying compounds useful for the diagnosis and treatment ofvarious PTP04-related diseases and conditions, for example cellproliferative disorders.

BACKGROUND OF THE INVENTION

The following description is provided to aid in understanding theinvention but is not admitted to be prior art to the invention.

Cellular signal transduction is a fundamental mechanism whereby externalstimuli that regulate diverse cellular processes are relayed to theinterior of cells. One of the key biochemical mechanisms of signaltransduction involves the reversible phosphorylation of proteins, whichenables regulation of the activity of mature proteins by altering theirstructure and function. The best characterized protein kinases ineukaryotes phosphorylate proteins on the alcohol moiety of serine,threonine and tyrosine residues. These kinases largely fall into twogroups, those specific for phosphorylating serines and threonines, andthose specific for phosphorylating tyrosines.

The phosphorylation state of a given substrate is also regulated by aclass of proteins responsible for removal of the phosphate group addedto a given substrate by a protein kinase. The protein phosphatases canalso be classified as being specific for either serine/threonine ortyrosine. The known enzymes can be divided into two groups—receptor andnon-receptor type proteins. Most receptor-type protein tyrosinephosphatases (RPTPs) contain two conserved catalytic tyrosinephosphatase domains each of which encompasses a segment of 240 aminoacid residues (Saito et al, Cell Growth and Diff. 2:59-65, 1991). TheRPTPs can be subclassified further based upon the amino acid sequencediversity of their extracellular domains (Saito, et al, supra; Krueger,et al, Proc. Natl. Acad. Sci. USA 89:7417-7421, 1992). Alignment ofprimary peptide sequences of both types of known PTPases shows somesequence consensus in catalytic domains and has made it possible toidentify cDNAs encoding proteins with tyrosine phosphate activity viathe polymerase chain reaction (PCR).

Many kinases and phosphatases are involved in regulatory cascadeswherein their substrates may include other kinases and phosphataseswhose activities are regulated by their phosphorylation state.Ultimately the activity of some downstream effector is modulated byphosphorylation resulting from activation of such a pathway.

Tyrosine phosphatases have been thought to be possible candidate cancercausing proteins. Inappropriate activity through overexpression ofRPTP-alpha, for example, has been associated with colon cancer (Pallen,et al, WO 94/01119, published Jan. 20, 1994). A need exists to identifyadditional proteins whose inappropriate activity may lead to cancer orother disorders so that pharmaceutical compounds for the treatment ofthose disorders might also be identified.

SUMMARY OF THE INVENTION

The present invention concerns PTP04 polypeptides, nucleic acidsencoding such polypeptides, cells, tissues and animals containing suchnucleic acids, antibodies to the polypeptides, assays utilizing thepolypeptides, and methods relating to all of the foregoing.

A first aspect of the invention features an isolated, enriched, orpurified nucleic acid molecule encoding a PTP04 polypeptide.

By “isolated” in reference to nucleic acid is meant a polymer of 14, 17,21 or more nucleotides conjugated to each other, including DNA or RNAthat is isolated from a natural source or that is synthesized. Theisolated nucleic acid of the present invention is unique in the sensethat it is not found in a pure or separated state in nature. Use of theterm “isolated” indicates that a naturally occurring sequence has beenremoved from its normal cellular (i.e., chromosomal) environment. Thus,the sequence may be in a cell-free solution or placed in a differentcellular environment. The term does not imply that the sequence is theonly nucleotide sequence present, but that it is essentially free (about90-95% pure at least) of non-nucleotide material naturally associatedwith it and thus is meant to be distinguished from isolated chromosomes.

By the use of the term “enriched” in reference to nucleic acid is meantthat the specific DNA or RNA sequence constitutes a significantly higherfraction (2-5 fold) of the total DNA or RNA present in the cells orsolution of interest than in normal or diseased cells or in the cellsfrom which the sequence was taken. This could be caused by a person bypreferential reduction in the amount of other DNA or RNA present, or bya preferential increase in the amount of the specific DNA or RNAsequence, or by a combination of the two. However, it should be notedthat “enriched” does not imply that there are no other DNA or RNAsequences present, just that the relative amount of the sequence ofinterest has been significantly increased.

The term “significant” here is used to indicate that the level ofincrease is useful to the person making such an increase, and generallymeans an increase relative to other nucleic acids of about at least 2fold, more preferably at least 5 to 10 fold or even more. The term alsodoes not imply that there is no DNA or RNA from other sources. The othersource DNA may, for example, comprise DNA from a yeast or bacterialgenome, or a cloning vector such as pUC19. This term distinguishes thesequence from naturally occurring enrichment events, such as viralinfection, or tumor type growths, in which the level of one mRNA may benaturally increased relative to other species of mRNA. That is, the termis meant to cover only those situations in which a person has intervenedto elevate the proportion of the desired nucleic acid.

It is also advantageous for some purposes that a nucleotide sequence bein purified form. The term “purified” in reference to nucleic acid doesnot require absolute purity (such as a homogeneous preparation);instead, it represents an indication that the sequence is relativelypurer than in the natural environment (compared to the natural levelthis level should be at least 2-5 fold greater, e.g., in terms ofmg/mL). Individual clones isolated from a cDNA library may be purifiedto electrophoretic homogeneity. The claimed DNA molecules obtained fromthese clones can be obtained directly from total DNA or from total RNA.The cDNA clones are not naturally occurring, but rather are preferablyobtained via manipulation of a partially purified naturally occurringsubstance (messenger RNA). The construction of a cDNA library from mRNAinvolves the creation of a synthetic substance (cDNA) and pureindividual cDNA clones can be isolated from the synthetic library byclonal selection of the cells carrying the cDNA library. Thus, theprocess which includes the construction of a cDNA library from mRNA andisolation of distinct cDNA clones yields an approximately 10⁶-foldpurification of the native message. Thus, purification of at least oneorder of magnitude, preferably two or three orders, and more preferablyfour or five orders of magnitude is expressly contemplated. The term isalso chosen to distinguish clones already in existence which may encodePTP04 but which have not been isolated from other clones in a library ofclones. Thus, the term covers clones encoding PTP04 which are isolatedfrom other non-PTP04 clones.

The term “nucleic acid molecule” describes a polymer ofdeoxyribonucleotides (DNA) or ribonucleotides (RNA). The nucleic acidmolecule may be isolated from a natural source by cDNA cloning orsubtractive hybridization or synthesized manually. The nucleic acidmolecule may be synthesized manually by the triester synthetic method orby using an automated DNA synthesizer.

The term “cDNA cloning” refers to hybridizing a small nucleic acidmolecule, a probe, to genomic cDNA. The probe hybridizes (binds) tocomplementary sequences of cDNA.

The term “complementary” describes two nucleotides that can formmultiple favorable interactions with one another. For example, adenineis complementary to thymine as they can form two hydrogen bonds.Similarly, guanine and cytosine are complementary since they can formthree hydrogen bonds. Thus if a nucleic acid sequence contains thefollowing sequence of bases, thymine, adenine, guanine and cytosine, a“complement” of this nucleic acid molecule would be a moleculecontaining adenine in the place of thymine, thymine in the place ofadenine, cytosine in the place of guanine, and guanine in the place ofcytosine. Because the complement can contain a nucleic acid sequencethat forms optimal interactions with the parent nucleic acid molecule,such a complement can bind with high affinity to its parent molecule.

The term “hybridize” refers to a method of interacting a nucleic acidsequence with a DNA or RNA molecule in solution or on a solid support,such as cellulose or nitrocellulose. If a nucleic acid sequence binds tothe DNA or RNA molecule with high affinity, it is said to “hybridize” tothe DNA or RNA molecule. The strength of the interaction between theprobing sequence and its target can be assessed by varying thestringency of the hybridization conditions. Under highly stringenthybrydization conditions only highly complementary nucleic acidsequences hybridize. Preferably, such conditions prevent hybridizationof nucleic acids having one or two mismatches out of 20 contiguousnucleotides.

Various low or high stringency hybridization conditions may be useddepending upon the specificity and selectivity desired. Stringency iscontrolled by varying salt or denaturant concentrations. Examples ofhybridization conditions are shown in the examples below. High stringentconditions may mean conditions that are at least as stringent as thefollowing: hybridization in 50% formamide, 5×SSC, 50 mM NaH₃PO₄, pH 6.8,0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5× Denhart solutionat 42° C. overnight; washing with 2×SSC, 0.1% SDS at 45° C.; and washingwith 0.2×SSC, 0.1% SDS at 45° C. Those skilled in the art will recognizehow such conditions can be varied to vary specificity and selectivity.

A PTP04 polypeptide can be encoded by a full-length nucleic acidsequence or any portion of the full-length nucleic acid sequence. Inpreferred embodiments the isolated nucleic acid comprises, consistsessentially of, or consists of a nucleic acid sequence set forth in SEQID NO:1, a nucleic acid sequence that hybridizes to the nucleic acidsequence set forth in SEQ ID NO:1 or a functional derivative (as definedbelow) of either. The nucleic acid may be isolated from a natural sourceby cDNA cloning or subtractive hybridization; the natural source may bemammalian (human) blood, semen, or tissue and the nucleic acid may besynthesized by the triester or other method or by using an automated DNAsynthesizer.

The term “mammalian” refers to such organisms as mice, rats, rabbits,goats, more preferably monkeys and apes, and most preferably humans.

In other preferred embodiments, the nucleic acid molecule of theinvention comprises a nucleotide sequence that (a) encodes a polypeptidehaving the full length amino acid sequence set forth in SEQ ID NO:2; (b)is the complement of the nucleotide sequence of (a); (c) hybridizesunder highly stringent conditions to the nucleotide molecule of (a) andencodes a naturally occurring PTP04 polypeptide; (d) encodes a PTP04polypeptide having the full length amino acid sequence of the sequenceset forth in SEQ ID NO:2, except that it lacks one or more of thefollowing segments of amino acid residues: 1-48, 49-294, 295-807 of SEQID NO:2; (e) is the complement of the nucleotide sequence of (d); (f)encodes a polypeptide having the amino acid sequence set forth in SEQ IDNO:2 from amino acid residues 1-48, 49-294, 295-807 of SEQ ID NO:2; (g)is the complement of the nucleotide sequence of (f); (h) encodes apolypeptide having the full length amino acid sequence set forth in SEQID NO:2, except that it lacks one or more of the domains selected fromthe group consisting of a signal peptide, an extracellular region, atransmembrane domain, a cytoplasmic domain, and a catalytic domain; or(i) is the complement of the nucleotide sequence of (h). The nucleicacid molecule of the invention is isolated, enriched, or purified from,preferably, a mammal, or most preferably from a human.

In yet other preferred embodiments the nucleic acid is an isolatedconserved or unique region, for example those useful for the design ofhybridization probes to facilitate identification and cloning ofadditional polypeptides, or for the design of PCR probes to facilitatecloning of additional polypeptides.

By “conserved nucleic acid regions”, it is meant regions present on twoor more nucleic acids encoding a PTP04 polypeptide, to which aparticular nucleic acid sequence can hybridize under lower stringencyconditions. Examples of lower stringency conditions suitable forscreening for nucleic acids encoding PTP04 polypeptides are provided inAbe, et al. J. Biol. Chem. 19:13361 (1992) (hereby incorporated byreference herein in its entirety, including any drawings). Preferably,conserved regions differ by no more than 5 out of 20 continguousnucleotides.

By “unique nucleic acid region” it is meant a sequence present in a fulllength nucleic acid coding for a PTP04 polypeptide that is not presentin a sequence coding for any other known naturally occurringpolypeptide. Such regions preferably comprise 14, 17, 21 or morecontiguous nucleotides present in the full length nucleic acid encodinga PTP04 polypeptide. In particular, a unique nucleic acid region ispreferably of human origin. In yet another aspect, the invention relatesto a nucleic acid vector comprising a nucleic acid molecule encoding aPTP04 polypeptide and a promoter element effective to initiatetranscription in a host cell.

The term “nucleic acid vector” relates to a single or double strandedcircular nucleic acid molecule that can be transfected or transformedinto cells and replicate independently or within the host cell genome. Acircular double stranded nucleic acid molecule can be cut and therebylinearized upon treatment with restriction enzymes. An assortment ofvectors, restriction enzymes, and the knowledge of the nucleotidesequences that the restriction enzymes operate upon are readilyavailable to those skilled in the art. A nucleic acid molecule of theinvention can be inserted into a vector by cutting the vector withrestriction enzymes and ligating the two pieces together.

Many techniques are available to those skilled in the art to facilitatetransformation or transfection of the expression construct into aprokaryotic or eukaryotic organism. The terms “transformation” and“transfection” refer to methods of inserting an expression constructinto a cellular organism. These methods involve a variety of techniques,such as treating the cells with high concentrations of salt, an electricfield, or detergent, to render the host cell outer membrane or wallpermeable to nucleic acid molecules of interest.

The term “promoter element” describes a nucleotide sequence that isincorporated into a vector that, once inside an appropriate cell, canfacilitate transcription factor and/or polymerase binding and subsequenttranscription of portions of the vector DNA into mRNA. The promoterelement precedes the 5′ end of the PTP04 nucleic acid molecule such thatthe latter is transcribed into mRNA. Host cell machinery then translatesmRNA into a polypeptide.

Those skilled in the art would recognize that a nucleic acid vector cancontain many other nucleic acid elements besides the promoter elementand the PTP04 nucleic acid molecule. These other nucleic acid elementsinclude, but are not limited to, origins of replication, ribosomalbinding sites, nucleic acid sequences encoding drug resistance enzymesor amino acid metabolic enzymes, and nucleic acid sequences encodingsecretion signals, periplasm or peroxisome localization signals, orsignals useful for polypeptide purification.

The invention also features a nucleic acid probe for the detection of anucleic acid encoding a PTP04 polypeptide in a sample.

The term “nucleic acid probe” refers to a nucleic molecule that iscomplementary to and can bind a nucleic acid sequence encoding the aminoacid sequence substantially similar to that set forth in SEQ ID NO:2.

The nucleic acid probe contains nucleic acid that will hybridizespecifically to a sequence of at least 14, preferably 17, 20 or 22,continguous nucleotides set forth in SEQ ID NO:1 or a functionalderivative thereof. The probe is preferably at least 14, 17 or morebases in length and selected to hybridize specifically to a uniqueregion of a PTP04 endocing nucleic acid.

In preferred embodiments the nucleic acid probe hybridizes to nucleicacid encoding at least 14 contiguous amino acids of the full-lengthsequence set forth in SEQ ID NO:1 or a functional derivative thereof.Various low or high stringency hybridization conditions may be useddepending upon the specificity and selectivity desired. Under highlystringent hybridization conditions only highly complementary nucleicacid sequences hybridize. Preferably, such conditions preventhybridization of nucleic acids having 1 or 2 mismatches out of 20contiguous nucleotides.

Methods for using the probes include detecting the presence or amount ofPTP04 RNA in a sample by contacting the sample with a nucleic acid probeunder conditions such that hybridization occurs and detecting thepresence or amount of the probe bound to PTP04 RNA. The nucleic acidduplex formed between the probe and a nucleic acid sequence coding for aPTP04 polypeptide may be used in the identification of the sequence ofthe nucleic acid detected (for example see, Nelson et al., inNonisotopic DNA Probe Techniques, p. 275 Academic Press, San Diego(Kricka, ed., 1992) hereby incorporated by reference herein in itsentirety, including any drawings). Kits for performing such methods maybe constructed to include a container means having disposed therein anucleic acid probe.

Another feature of the invention is a nucleic acid molecule as set forthin SEQ ID NO:1 or fragments thereof, comprising one or more regions thatencode a PTP04 polypeptide or a PTP04 domain polypeptide, where thePTP04 polypeptide or the PTP04 domain polypeptide is fused to anon-PTP04 polypeptide. Such fused polypeptides include, for example, butare not limited to, a GST-fusion protein.

The invention also features recombinant nucleic acid, preferably in acell or an organism. The recombinant nucleic acid may contain a sequenceset forth in SEQ ID NO:1 or a functional derivative thereof and a vectoror a promoter effective to initiate transcription in a host cell. Therecombinant nucleic acid can alternatively contain a transcriptionalinitiation region functional in a cell, a sequence complimentary to anRNA sequence encoding a PTP04 polypeptide and a transcriptionaltermination region functional in a cell.

Another aspect of the invention relates to a recombinant cell or tissuecomprising a nucleic acid molecule encoding a PTP04 polypeptide. Therecombinant cell may comprise a nucleic acid molecule encoding either aPTP04 polypeptide; a PTP04 domain polypeptide; or a PTP04 polypeptide orPTP04 domain polypeptide fused to a non-PTP04 polypeptide.

The term “recombinant organism” refers to an organism that has a newcombination of genes or nucleic acid molecules. A new combination ofgenes or nucleic acid molecules can be introduced to an organism using awide array of nucleic acid manipulation techniques available to thoseskilled in the art.

The term “organism” relates to any living being comprised of a least onecell. An organism can be as simple as one eukaryotic cell or as complexas a mammal. Therefore, a recombinant organism can also be a recombinantcell, which may be a eukaryotic or a prokaryotic organism.

The term “eukaryote” refers to an organism comprised of cells thatcontain a nucleus. Eukaryotes are differentiated from “prokaryotes”which do not have a nucleus and lack other cellular structures found ineukaryotes, such as mitochondria and endoplasmic reticulum. Prokaryotesinclude unicellular organisms, such as bacteria, while eukaryotes arerepresented by yeast, invertebrates, and vertebrates.

The recombinant cell can harbor a nucleic acid vector that isextragenomic. The term “extragenomic” refers to a nucleic acid vectorwhich does not insert into the cell genome. Many nucleic acid vectorsare designed with their own origins of replication allowing them toutilize the recombinant cell replication machinery to copy and propagatethe vector nucleic acid sequence. These vectors are small enough thatthey are not likely to harbor nucleic acid sequences homologous togenomic sequences of the recombinant cell. Thus these vectors replicateindependently of the host genome and do not recombine with or integrateinto the genome.

A recombinant cell can harbor a portion of a nucleic acid vector in anintragenomic fashion. The term “intragenomic” defines a nucleic acidconstruct that is incorporated within the cell genome. Multiple nucleicacid vectors available to those skilled in the art contain nucleic acidsequences that are homologous to nucleic acid sequences in a particularorganism's genomic DNA. These homologous sequences will result inrecombination events that integrate portions of the vector into thegenomic DNA. Those skilled in the art can control which nucleic acidsequences of the vector are integrated into the cell genome by flankingthe portion to be incorporated into the genome with homologous sequencesin the vector.

Another aspect of the invention features an isolated, enriched, orpurified PTP04 polypeptide.

By “PTP04 polypeptide” it is meant an amino acid sequence substantiallysimilar to the sequence shown in SEQ ID NO:2, or fragments thereof. Asequence that is substantially similar will preferably have at least 90%identity (more preferably at least 95% and most preferably 99-100%) tothe sequence of SEQ ID NO:2.

The PTP04 polypeptides of the present invention preferably have asubstantially similar biological activity to the protein encoded by thefull length nucleic acid sequence set forth in SEQ ID NO:1 or to theproteins with amino acid sequence set forth in SEQ ID NO:2. By“biological activity” it is meant an activity of the PTP04 protein in acell. The biological activity of the PTP04 is related to some of theactivities of the cell which include, but are not limited to, cellproliferation motogenesis, metastasis, tumor escape, cell adhesion,transformation, or apoptosis.

The PTP04 polypeptides of the present invention preferably have asubstantially similar biological activity to the protein encoded by thefull length nucleic acid sequence set forth in SEQ ID NO:1 or to theproteins with amino acid sequence set forth in SEQ ID NO:2. By“biological activity” it is meant an activity of the PTP04 protein in acell. The biological activity of the PTP04 is related to some of theactivities of the cell which include, but are not limited to, cellproliferation motogenesis, metastasis, tumor escape, cell adhesion,transformation, or apoptosis.

By “identity” is meant a property of sequences that measures theirsimilarity or relationship. Identity is measured by dividing the numberof identical residues in the two sequences by the total number ofresidues and multiplying the product by 100. Thus, two copies of exactlythe same sequence have 100% identity, but sequences that are less highlyconserved and have deletions, additions, or replacements have a lowerdegree of identity. Those skilled in the art will recognize that severalcomputer programs are available for determining sequence identity.

By “isolated” in reference to a polypeptide is meant a polymer of 6, 12,18 or more amino acids conjugated to each other, including polypeptidesthat are isolated from a natural source or that are synthesized. Theisolated polypeptides of the present invention are unique in the sensethat they are not found in a pure or separated state in nature. Use ofthe term “isolated” indicates that a naturally occurring sequence hasbeen removed from its normal cellular environment. Thus, the sequencemay be in a cell-free solution or placed in a different cellularenvironment. The term does not imply that the sequence is the only aminoacid chain present, but that it is essentially free (about 90-95% pureat least) of material naturally associated with it.

By the use of the term “enriched” in reference to a polypeptide it ismeant that the specific amino acid sequence constitutes a significantlyhigher fraction (2-5 fold) of the total of amino acid sequences presentin the cells or solution of interest than in normal or diseased cells orin the cells from which the sequence was taken. This could be caused bya person by preferential reduction in the amount of other amino acidsequences present, or by a preferential increase in the amount of thespecific amino acid sequence of interest, or by a combination of thetwo. However, it should be noted that “enriched” does not imply thatthere are no other amino acid sequences present, just that the relativeamount of the sequence of interest has been significantly increased.

The term “significant” here is used to indicate that the level ofincrease is useful to the person making such an increase, and generallymeans an increase relative to other amino acid sequences of about atleast 2 fold, more preferably at least 5 to 10 fold or even more. Theterm also does not imply that there are no amino acid sequences fromother sources. The other source amino acid may, for example, compriseamino acid sequences encoded by a yeast or bacterial genome, or acloning vector such as pUC19. The term is meant to cover only thosesituations in which a person has intervened to elevate the proportion ofthe desired nucleic acid sequence.

It is also advantageous for some purposes that an amino acid sequence bein purified form. The term “purified” in reference to a polypeptide doesnot require absolute purity (such as a homogeneous preparation);instead, it represents an indication that the sequence is relativelypurer than in the natural environment (compared to the natural levelthis level should be at least 2-5 fold greater, e.g., in terms ofmg/mL). Purification of at least one order of magnitude, preferably twoor three orders, and more preferably four or five orders of magnitude isexpressly contemplated. The substance is preferably free ofcontamination at a functionally significant level, for example 90%, 95%,or 99% pure.

In another aspect the invention features an isolated, enriched, orpurified PTP04 polypeptide fragment.

By “a PTP04 polypeptide fragment” it is meant an amino acid sequencethat is less than the full-length PTP04 amino acid sequence shown in SEQID NO:2. Examples of fragments include PTP04 domains, PTP04 mutants andPTP04-specific epitopes.

By “a PTP04 domain” it is meant a portion of the PTP04 polypeptidehaving homology to amino acid sequences from one or more known proteinswherein the sequence predicts some common function, interaction oractivity. Well known examples of domains are the SH2 (Src Homology 2)domain (Sadowski, et al., Mol. Cell. Biol. 6:4396, 1986; Pawson andSchlessinger, Curr. Biol. 3:434, 1993), the SH3 domain (Mayer, et al.,Nature 332:272, 1988; Pawson and Schlessinger, Curr. Biol. 3:434, 1993),and pleckstrin (PH) domain (Ponting, TIBS 21:245, 1996; Haslam, et al.,Nature 363:309, 1993), all of which are domains that mediateprotein:protein interaction, and the kinase catalytic domain (Hanks andHunter, FASEB J 9:576-595, 1995). Computer programs designed to detectsuch homologies are well known in the art. The relative homology is atleast 20%, more preferably at least 30% and most preferably at least35%.

By a “PTP04 mutant” it is meant a PTP04 polypeptide which differs fromthe native sequence in that one or more amino acids have been changed,added or deleted. Changes in amino acids may be conservative ornon-conservative. By “conservative” it is meant the substitution of anamino acid for one with similar properties such as charge,hydrophobicity, structure, etc. Examples of polypeptides encompassed bythis term include, but are not limited to, (1) chimeric proteins whichcomprise a portion of a PTP04 polypeptide sequence fused to a non-PTP04polypeptide sequence, for example a polypeptide sequence ofhemagglutinin (HA), (2) PTP04 proteins lacking a specific domain, forexample the catalytic domain, and (3) PTP04 proteins having a pointmutation. A PTP04 mutant will retain some useful function such as, forexample, binding to a natural binding partner, catalytic activity, orthe ability to bind to a PTP04 specific antibody (as defined below).

By “PTP04-specific epitope” it is meant a sequence of amino acids thatis both antigenic and unique to PTP04. PTP04-specific epitope can beused to produce PTP04-specific antibodies, as more fully describedbelow. Particularly preferred epitopes are shown in Example 4 below.

By “recombinant PTP04 polypeptide” it is meant to include a polypeptideproduced by recombinant DNA techniques such that it is distinct from anaturally occurring polypeptide either in its location (e.g., present ina different cell or tissue than found in nature), purity or structure.Generally, such a recombinant polypeptide will be present in a cell inan amount different from that normally observed in nature.

The polypeptide of the invention comprises an amino acid sequence having(a) the full length amino acid sequence set forth in SEQ ID NO:2; (b)the full length amino acid sequence of the sequence set forth in SEQ IDNO:2, except that it lacks one or more of the following segments ofamino acid residues: 1-48, 49-294, 295-807 of SEQ ID NO:2; (c) the aminoacid sequence set forth in SEQ ID NO:2 from amino acid residues, 1-48,49-294, 295-807 of SEQ ID NO:2; or (d) the full length amino acidsequence set forth in SEQ ID NO:2 except that it lacks one or more ofthe domains selected from the group consisting of a signal peptide, anextracellular region, a transmembrane domain, a cytoplasmic domain, anda catalytic domain.

In yet another aspect the invention features an antibody (e.g., amonoclonal or polyclonal antibody) having specific binding affinity to aPTP04 polypeptide or PTP04 polypeptide fragment. By “specific bindingaffinity” is meant that the antibody binds to target (PTP04)polypeptides with greater affinity than it binds to other polypeptidesunder specified conditions. Antibodies having specific binding affinityto a PTP04 polypeptide may be used in methods for detecting the presenceand/or amount of a PTP04 polypeptide in a sample by contacting thesample with the antibody under conditions such that an immunocomplexforms and detecting the presence and/or amount of the antibodyconjugated to the PTP04 polypeptide. Diagnostic kits for performing suchmethods may be constructed to include a first container containing theantibody and a second container having a conjugate of a binding partnerof the antibody and a label, such as, for example, a radioisotope. Thediagnostic kit may also include notification of an FDA approved use andinstructions therefor.

The term “polyclonal” refers to antibodies that are heterogenouspopulations of antibody molecules derived from the sera of animalsimmunized with an antigen or an antigenic functional derivative thereof.For the production of polyclonal antibodies, various host animals may beimmunized by injection with the antigen. Various adjuvants may be usedto increase the immunological response, depending on the host species.

“Monoclonal antibodies” are substantially homogenous populations ofantibodies to a particular antigen. They may be obtained by anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. Monoclonal antibodies may be obtainedby methods known to those skilled in the art. See, for example, Kohler,et al., Nature 256:495-497 (1975), and U.S. Pat. No. 4,376,110.

The term “antibody fragment” refers to a portion of an antibody, oftenthe hypervariable region and portions of the surrounding heavy and lightchains, that displays specific binding affinity for a particularmolecule. A hypervariable region is a portion of an antibody thatphysically binds to the polypeptide target.

In another aspect the invention features a hybridoma which produces anantibody having specific binding affinity to a PTP04 polypeptide. By“hybridoma” is meant an immortalized cell line which is capable ofsecreting an antibody, for example a PTP04 antibody. In preferredembodiments the PTP04 antibody comprises a sequence of amino acids thatis able to specifically bind a PTP04 polypeptide.

The invention features a method for identifying human cells containing aPTP04 polypeptide or a related sequence. The method involves identifyingthe novel polypeptide in human cells using techniques that are routineand standard in the art, such as those described herein for identifyingPTP04 (e.g., cloning, Southern or Northern blot analysis, in situhybridization, PCR amplification, etc.).

The invention also features methods of screening cells for naturalbinding partners of PTP04 polypeptides. By “natural binding partner” itis meant a protein that interacts with PTP04. Binding partners includeligands, agonists, antagonists and downstream signaling molecules suchas adaptor proteins and may be identified by techniques well known inthe art such as co-immunoprecipitation or by using, for example, atwo-hybrid screen. (Fields and Song, U.S. Pat. No. 5,283,173, issuedFeb. 1, 1994 and, incorporated be reference herein.) The presentinvention also features the purified, isolated or enriched versions ofthe polypeptides identified by the methods described above.

In another aspect, the invention provides a method for identifying asubstance capable of modulating PTP04 activity comprising the steps of(a) contacting a PTP04 polypeptide with a test substance; and (b)determining whether the substance alters the activity of saidpolypeptide.

The invention also features another method of identifying substancescapable of modulating the function of a PTP04 polypeptide. The methodcomprises the following steps: (a) expressing a PTP04 polypeptide incells; (b) adding a compound to the cells; and (c) monitoring a changeor an absence of a change in cell phenotype, cell proliferation,catalytic activity of the PTP04 polypeptide, and binding a naturalbinding partner.

The term “compound” includes small organic molecules including, but notlimited to, oxindolinones, quinazolines, tyrphostins, quinoxalines, andthose contained within extracts from natural sources. Examples of suchcompounds are included in section XII, below.

The term “function” refers to the cellular role of a serine-threonineprotein kinase. The serine-threonine protein kinase family includesmembers that regulate many steps in signaling cascades, includingcascades controlling cell growth, migration, differentiation, geneexpression, muscle contraction, glucose metabolism, cellular proteinsynthesis, and regulation of the cell cycle.

The term “modulates” refers to the ability of a compound to alter thefunction of a protein kinase. A modulator preferably activates thecatalytic activity of a protein kinase, more preferably activates orinhibits the catalytic activity of a protein kinase depending on theconcentration of the compound exposed to the protein kinase, or mostpreferably inhibits the catalytic activity of a protein kinase.

The term “catalytic activity,” in the context of the invention, definesthe ability of a protein kinase to phosphorylate a substrate. Catalyticactivity can be measured, for example, by determining the amount of asubstrate converted to a product as a function of time. Phosphorylationof a substrate occurs at the active-site of a protein kinase. Theactive-site is normally a cavity in which the substrate.

The term “substrate” as used herein refers to a molecule that isphoshorylated by or directly interacts with the protein kinase. Thesubstrate is preferably a peptide and more preferably a protein. Forexample, in relation to the protein kinase RAF, preferred substrates areMEK and the MEK substrate MAPK.

The term “activates” refers to increasing the cellular function of aprotein kinase. The protein kinase function is preferably theinteraction with a natural binding partner or catalytic activity.

The term “inhibit” refers to decreasing the cellular function of aprotein kinase. The protein kinase function is preferably theinteraction with a natural binding partner or catalytic activity.

The term “modulates” also refers to altering the function of a proteinkinase by increasing or decreasing the probability that a complex formsbetween a protein kinase and a natural binding partner. A modulatorpreferably increases the probability that such a complex forms betweenthe protein kinase and the natural binding partner, more preferably increases or decreases the probability that a complex forms between theprotein kinase and the natural binding partner depending on theconcentration of the compound exposed to the protein kinase, and mostpreferably decreases the probability that a complex forms between theprotein kinase and the natural binding partner.

The term “complex” refers to an assembly of at least two molecules boundto one another. Signal transduction complexes often contain at least twoprotein molecules bound to one another, either transiently or insuccession. For instance, a receptor protein tyrosine kinase, GRB2, SOS,and RAF sequentially interact in response to a mitogenic ligand.

The term “expressing” as used herein refers to the production of a PTP04polypeptide from a nucleic acid vector containing a PTP04 gene within acell. The nucleic acid vector is transfected into cells using well knowntechniques in the art as described herein.

The term “adding” as used herein refers to administering a solutioncomprising a compound to the medium bathing cells. The solutioncomprising the compound can also comprise an agent, such as dimethylsulfoxide, which facilitates the uptake of the compound into the cells.

The term “monitoring” refers to observing the effect of adding thecompound to the cells of the method. The effect can be manifested in achange in cell phenotype, cell proliferation, protein kinase catalyticactivity, or in the interaction between a protein kinase and a naturalbinding partner.

The term “cell phenotype” refers to the outward appearance of a cell ortissue or the function of the cell or tissue. Examples of cell or tissuephenotype are cell size (reduction or enlargement), cell proliferation(increased or decreased numbers of cells), cell differentiation (achange or absence of a change in cell shape), cell survival, apoptosis(cell death), or the utilization of a metabolic nutrient (e.g., glucoseuptake). Change or the absence of change in cell phenotype is readilymeasured by techniques known in the art.

The term “cell proliferation” refers to the rate at which a group ofcells divides. The number of cells growing in a vessel can bequantitated by a person skilled in the art when that person visuallycounts the number of cells in a defined area using a common lightmicroscope. Alternatively, cell proliferation rates can be quantitatedby laboratory apparatus that optically measure the density of cells inan appropriate medium.

The method can utilize any of the molecules disclosed in the invention.These molecules include nucleic acid molecules encoding PTP04polypeptides, nucleic acid vectors, recombinant cells, polypeptides, orantibodies of the invention.

In a preferred embodiment, the invention provides a method for treatingor preventing an abnormal condition by administering a compound which isa modular of PTP04 function in vitro. The abnormal condition preferablyinvolves abnormality in PTP04 signal transduction pathway, and mostpreferably is cancer. Such compounds preferably show positive results inone or more in vitro assays for an activity corresponding to treatmentof the disease or disorder in question (such as the assays described inExample 6 below). Examples of substances that can be screened forfavorable activity are provided in section XII below.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a comparison between the amino acid sequence of huma PTP04and the amino acid sequence of the protein to which it is most closelyrelated, murine ZPEP. The relative homology between the two(approximately 70%) suggests that the two proteins are members of thesame PTP family but are not species orthologs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the isolation and characterization of anew protein which we have called PTP04, nucleotide sequences encodingPTP04, various products and assay methods that can be used to identifycompounds useful for the diagnosis and treatment of various PTP04related diseases and conditions, for example cancer. Polypeptidesderived from PTP04 and nucleic acids encoding such polypeptides may beproduced using well known and standard synthesis techniques when giventhe sequences presented herein.

PTP04 is a tyrosine phosphatase with an apparent molecular weight ofapproximately 100 kDa. Primary sequence analysis shows that PTP04 iscomprised of three domains: an N-terminal domain, a catalytic domain,and a C-terminal domain. The lack of a hydrophobic stretch of aminoacids generally characterized as a transmembrane region indicates thatPTP04 is a non-receptor tyrosine phosphatase.

The full-length PTP04 was originally isolated from a human leukemia cellline. Subsequent expression analysis of both normal tissues and cancercell lines, shown in detail below, revealed that PTP04 is expressed inhuman thymus and has very low expression in other normal cells but issignificantly overexpressed in a number of tumors, particularly inleukemias and lymphomas. This suggests that PTP04 plays an importantrole in the growth and persistence of these cancers.

The polypeptide and nucleotide sequences of the invention can be used,therefore, to identify modulators of cell growth and survival which areuseful in developing therapeutics for various cell proliferativedisorders and conditions, and in particular cancers related toinappropriate PTP04 activity. Assays to identify compounds that actintracellularly to enhance or inhibit PTP04 activity can be developed bycreating genetically engineered cell lines that express PTP04 nucleotidesequences, as is more fully discussed below.

I. Nucleic Acids Encoding PTP04 Polypeptides.

A first aspect of the invention features nucleic acid sequences encodinga PTP04 polypeptide. Included within the scope of this invention are thefunctional equivalents of the herein-described isolated nucleic acidmolecules. Functional equivalents or derivatives can be obtained inseveral ways. The degeneracy of the genetic code permits substitution ofcertain codons by other codons which specify the same amino acid andhence would give rise to the same protein. The nucleic acid sequence canvary substantially since, with the exception of methionine andtryptophan, the known amino acids can be coded for by more than onecodon. Thus, portions or all of the PTP04 gene could be synthesized togive a nucleic acid sequence significantly different from that shown inSEQ ID NO:1. The encoded amino acid sequence thereof would, however, bepreserved.

In addition, the nucleic acid sequence may comprise a nucleotidesequence which results from the addition, deletion or substitution of atleast one nucleotide to the 5′-end and/or the 3′-end of the nucleic acidformula shown in SEQ ID NO:1 or a derivative thereof. Any nucleotide orpolynucleotide may be used in this regard, provided that its addition,deletion or substitution does not alter the amino acid sequence of SEQID NO:2 which is encoded by the nucleotide sequence. For example, thepresent invention is intended to include any nucleic acid sequenceresulting from the addition of ATG as an initiation codon at the 5′-endof the PTP04 nucleic acid sequence or its functional derivative, or fromthe addition of TTA, TAG or TGA as a termination codon at the 3′-end ofthe inventive nucleotide sequence or its derivative. Moreover, thenucleic acid molecule of the present invention may, as necessary, haverestriction endonuclease recognition sites added to its 5′-end and/or3′-end.

Such functional alterations of a given nucleic acid sequence afford anopportunity to promote secretion and/or processing of heterologousproteins encoded by foreign nucleic acid sequences fused thereto. Allvariations of the nucleotide sequence of the PTP04 genes and fragmentsthereof permitted by the genetic code are, therefore, included in thisinvention.

Further, it is possible to delete codons or to substitute one or morecodons by codons other than degenerate codons to produce a structurallymodified polypeptide, but one which has substantially the same utilityor activity of the polypeptide produced by the unmodified nucleic acidmolecule. As recognized in the art, the two polypeptides arefunctionally equivalent, as are the two nucleic acid molecules whichgive rise to their production, even though the differences between thenucleic acid molecules are not related to degeneracy of the geneticcode.

Functional equivalents or derivatives of PTP04 can also be obtainedusing nucleic acid molecules encoding one or more functional domains ofthe PTP04 polypeptide. For example, the catalytic domain of PTP04functions as an enzymatic remover of phosphate molecules bound ontotyrosine amino acids and a nucleic acid sequence encoding the catalyticdomain alone or linked to other heterologous nucleic acid sequences canbe considered a functional derivative of PTP04. Other functional domainsof PTP04 include, but are not limited to, the proline-rich region withinthe N-terminal domain, and the C-terminal domain. Nucleic acid sequencesencoding these domains are shown in SEQ. ID NO.:1 as follows: N-terminaldomain 53-196; catalytic domain 197-934, C-terminal domain 935-2473.

II. A Nucleic Acid Probe for the Detection of PTP04.

A nucleic acid probe of the present invention may be used to probe anappropriate chromosomal or cDNA library by usual hybridization methodsto obtain another nucleic acid molecule of the present invention. Achromosomal DNA or cDNA library may be prepared from appropriate cellsaccording to recognized methods in the art (e.g. “Molecular Cloning: ALaboratory Manual”, second edition, edited by Sambrook, Fritsch, &Maniatis, Cold Spring Harbor Laboratory, 1989).

In the alternative, chemical synthesis is carried out in order to obtainnucleic acid probes having nucleotide sequences which correspond toN-terminal and C-terminal portions of the amino acid sequence of thepolypeptide of interest. Thus, the synthesized nucleic acid probes maybe used as primers in a polymerase chain reaction (PCR) carried out inaccordance with recognized PCR techniques, essentially according to PCRProtocols, “A Guide to Methods and Applications”, edited by Michael etal., Academic Press, 1990, utilizing the appropriate chromosomal or cDNAlibrary to obtain the fragment of the present invention.

One skilled in the art can readily design such probes based on thesequence disclosed herein using methods of computer alignment andsequence analysis known in the art (e.g. “Molecular Cloning: ALaboratory Manual”, second edition, edited by Sambrook, Fritsch, &Maniatis, Cold Spring Harbor Laboratory, 1989). The hybridization probesof the present invention can be labeled by standard labeling techniquessuch as with a radiolabel, enzyme label, fluorescent label,biotin-avidin label, chemiluminescence, and the like. Afterhybridization, the probes may be visualized using known methods.

The nucleic acid probes of the present invention include RNA as well asDNA probes and nucleic acids modified in the sugar, phosphate or eventhe base portion as long as the probe still retains the ability tospecifically hybridize under conditions as disclosed herein. Such probesare generated using techniques known in the art. The nucleic acid probemay be immobilized on a solid support. Examples of such solid supportsinclude, but are not limited to, plastics such as polycarbonate, complexcarbohydrates such as agarose and sepharose, acrylic resins, such aspolyacrylamide and latex beads, and nitrocellulose. Techniques forcoupling nucleic acid probes to such solid supports are well known inthe art.

The test samples suitable for nucleic acid probing methods of thepresent invention include, for example, cells or nucleic acid extractsof cells, or biological fluids. The sample used in the above-describedmethods will vary based on the assay format, the detection method andthe nature of the tissues, cells or extracts to be assayed.

Methods for preparing nucleic acid extracts of cells are well known inthe art and can be readily adapted in order to obtain a sample which iscompatible with the method utilized.

III. A Probe Based Method and Kit for Detecting PTP04.

One method of detecting the presence of PTP04 in a sample comprises (a)contacting the sample with the above-described nucleic acid probe, underconditions such that hybridization occurs, and (b) detecting thepresence of the probe bound to the nucleic acid molecule. One skilled inthe art would select the nucleic acid probe according to techniquesknown in the art as described above. Samples to be tested include butshould not be limited to RNA samples of human tissue.

A kit for detecting the presence of PTP04 in a sample comprises at leastone container having disposed therein the above-described nucleic acidprobe. The kit may further comprise other containers comprising one ormore of the following: wash reagents and reagents capable of detectingthe presence of bound nucleic acid probe. Examples of detection reagentsinclude, but are not limited to radiolabelled probes, enzymaticlylabeled probes (horseradish peroxidase, alkaline phosphatase), andaffinity labeled probes (biotin, avidin, or steptavidin).

In detail, a compartmentalized kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers or strips of plastic or paper. Suchcontainers allow the efficient transfer of reagents from one compartmentto another compartment such that the samples and reagents are notcross-contaminated and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the probe or primers used in the assay,containers which contain wash reagents (such as phosphate bufferedsaline, Tris-buffers, and the like), and containers which contain thereagents used to detect the hybridized probe, bound antibody, amplifiedproduct, or the like. One skilled in the art will readily recognize thatthe nucleic acid probes described in the present invention can readilybe incorporated into one of the established kit formats which are wellknown in the art with or without a set of instructions concerning theuse of such reagents in an assay.

IV. DNA Constructs Comprising a PTP04 Nucleic Acid Molecule and CellsContaining These Constructs.

The present invention also relates to a recombinant DNA moleculecomprising, 5′ to 3′, a promoter effective to initiate transcription ina host cell and the above-described nucleic acid molecules. In addition,the present invention relates to a recombinant DNA molecule comprising avector and a nucleic acid molecule described herein. The presentinvention also relates to a nucleic acid molecule comprising atranscriptional region functional in a cell, a sequence complimentary toan RNA sequence encoding an amino acid sequence corresponding to a PTP04polypeptide or functional derivative, and a transcriptional terminationregion functional in said cell. The above-described molecules may beisolated and/or purified DNA molecules.

The present invention also relates to a cell or organism that contains aPTP04 nucleic acid molecule as described herein and thereby is capableof expressing a peptide. The polypeptide may be purified from cellswhich have been altered to express the polypeptide. A cell is said to be“altered to express a desired polypeptide” when the cell, throughgenetic manipulation, is made to produce a protein which it normallydoes not produce or which the cell normally produces at lower levels.One skilled in the art can readily adapt procedures for introducing andexpressing either genomic, cDNA, or synthetic sequences into eithereukaryotic or prokaryotic cells.

A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are “operably linked” to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene sequence expression. Theprecise nature of the regulatory regions needed for gene sequenceexpression may vary from organism to organism, but will in generalinclude a promoter region which, in prokaryotes, contains both thepromoter (which directs the initiation of RNA transcription) as well asthe DNA sequences which, when transcribed into RNA, will signalsynthesis initiation. Such regions will normally include those5′-non-coding sequences involved with initiation of transcription andtranslation, such as the TATA box, capping sequence, CAAT sequence, andthe like.

If desired, the non-coding region 3′ to the sequence encoding a PTP04gene may be obtained by the above-described cloning methods. This regionmay be retained for its transcriptional termination regulatorysequences, such as termination and polyadenylation. Thus, by retainingthe 3′-region naturally contiguous to the DNA sequence encoding a PTP04gene, the transcriptional termination signals may be provided. Where thetranscriptional termination signals are not satisfactorily functional inthe expression host cell, then a 3′ region functional in the host cellmay be substituted.

Two DNA sequences (such as a promoter region sequence and a PTP04sequence) are said to be operably linked if the nature of the linkagebetween the two DNA sequences does not (1) result in the introduction ofa frame-shift mutation, (2) interfere with the ability of the promoterregion sequence to direct the transcription of a PTP04 gene sequence, or(3) interfere with the ability of the a PTP04 gene sequence to betranscribed by the promoter region sequence. Thus, a promoter regionwould be operably linked to a DNA sequence if the promoter were capableof effecting transcription of that DNA sequence. Thus, to express aPTP04 gene, transcriptional and translational signals recognized by anappropriate host are necessary.

The present invention encompasses the expression of a PTP04 gene (or afunctional derivative thereof) in either prokaryotic or eukaryoticcells. Prokaryotic hosts are, generally, very efficient and convenientfor the production of recombinant proteins and are, therefore, one typeof preferred expression system for a PTP04 gene. Prokaryotes mostfrequently are represented by various strains of E. coli. However, othermicrobial strains may also be used, including other bacterial strains.

In prokaryotic systems, plasmid vectors that contain replication sitesand control sequences derived from a species compatible with the hostmay be used. Examples of suitable plasmid vectors may include pBR322,pUC118, pUC119 and the like; suitable phage or bacteriophage vectors mayinclude lgt10, lgt11 and the like; and suitable virus vectors mayinclude pMAM-neo, pKRC and the like. Preferably, the selected vector ofthe present invention has the capacity to replicate in the selected hostcell.

Recognized prokaryotic hosts include bacteria such as E. coli and thosefrom genera such as Bacillus, Streptomyces, Pseudomonas, Salmonella,Serratia, and the like. However, under such conditions, the polypeptidewill not be glycosylated. The prokaryotic host must be compatible withthe replicon and control sequences in the expression plasmid.

To express PTP04 (or a functional derivative thereof) in a prokaryoticcell, it is necessary to operably link a PTP04 sequence to a functionalprokaryotic promoter. Such promoters may be either constitutive or, morepreferably, regulatable (i.e., inducible or derepressible). Examples ofconstitutive promoters include the int promoter of bacteriophage 1, thebla promoter of the b-lactamase gene sequence of pBR322, and the CATpromoter of the chloramphenicol acetyl transferase gene sequence ofpPR325, and the like. Examples of inducible prokaryotic promotersinclude the major right and left promoters of bacteriophage l (P_(L) andP_(R)), the trp, recA, lacZ, lacI, and gal promoters of E. coli, thea-amylase (Ulmanen et al., J. Bacteriol. 162:176-182, 1985) and thesigma-28-specific promoters of B. subtilis (Gilman et al., Gene Sequence32:11-20(1984)), the promoters of the bacteriophages of Bacillus(Gryczan, In: The Molecular Biology of the Bacilli, Academic Press,Inc., NY (1982)), and Streptomyces promoters (Ward et al., Mol. Gen.Genet. 203:468-478, 1986). Prokaryotic promoters are reviewed by Glick(J. Ind. Microbiot. 1:277-282, 1987); Cenatiempo (Biochimie 68:505-516,1986); and Gottesman (Ann. Rev. Genet. 18:415-442, 1984).

Proper expression in a prokaryotic cell also requires the presence of aribosome binding site upstream of the gene sequence-encoding sequence.Such ribosome binding sites are disclosed, for example, by Gold et at.(Ann. Rev. Microbiol. 35:365-404, 1981). The selection of controlsequences, expression vectors, transformation methods, and the like, aredependent on the type of host cell used to express the gene.

As used herein, “cell”, “cell line”, and “cell culture” may be usedinterchangeably and all such designations include progeny. Thus, thewords “transformants” or “transformed cells” include the primary subjectcell and cultures derived therefrom, without regard to the number oftransfers. It is also understood that all progeny may not be preciselyidentical in DNA content, due to deliberate or inadvertent mutations.However, as defined, mutant progeny have the same functionality as thatof the originally transformed cell.

Host cells which may be used in the expression systems of the presentinvention are not strictly limited, provided that they are suitable foruse in the expression of the PTP04 peptide of interest. Suitable hostsmay often include eukaryotic cells. Preferred eukaryotic hosts include,for example, yeast, fungi, insect cells, mammalian cells either in vivo,or in tissue culture. Mammalian cells which may be useful as hostsinclude HeLa cells, cells of fibroblast origin such as VERO, 3T3 orCHO-K1, or cells of lymphoid origin (such as 32D cells) and theirderivatives. Preferred mammalian host cells include SP2/0 and J558L, aswell as neuroblastoma cell lines such as IMR 332 and PC12 which mayprovide better capacities for correct post-translational processing.

In addition, plant cells are also available as hosts, and controlsequences compatible with plant cells are available, such as thecauliflower mosaic virus 35S and 19S, and nopaline synthase promoter andpolyadenylation signal sequences. Another preferred host is an insectcell, for example the Drosophila larvae. Using insect cells as hosts,the Drosophila alcohol dehydrogenase promoter can be used. Rubin,Science 240:1453-1459, 1988). Alternatively, baculovirus vectors can beengineered to express large amounts of PTP04 in insects cells (Jasny,Science 238:1653, 1987); Miller et al., In: Genetic Engineering (1986),Setlow, J. K., et al., eds., Plenum, Vol. 8, pp. 277-297).

Any of a series of yeast gene sequence expression systems can beutilized which incorporate promoter and termination elements from theactively expressed gene sequences coding for glycolytic enzymes areproduced in large quantities when yeast are grown in mediums rich inglucose. Known glycolytic gene sequences can also provide very efficienttranscriptional control signals. Yeast provides substantial advantagesin that it can also carry out post-translational peptide modifications.A number of recombinant DNA strategies exist which utilize strongpromoter sequences and high copy number of plasmids which can beutilized for production of the desired proteins in yeast. Yeastrecognizes leader sequences on cloned mammalian gene sequence productsand secretes peptides bearing leader sequences (i.e., pre-peptides). Fora mammalian host, several possible vector systems are available for theexpression of PTP04.

A particularly preferred yeast expression system is that utilizingSchizosaccharmocyces pombe. This system is useful for studying theactivity of members of the Src family (Superti-Furga, et al., EMBO J.12:2625, 1993) and other NR-TKs.

A wide variety of transcriptional and translational regulatory sequencesmay be employed, depending upon the nature of the host. Thetranscriptional and translational regulatory signals may be derived fromviral sources, such as adenovirus, bovine papilloma virus,cytomegalovirus, simian virus, or the like, where the regulatory signalsare associated with a particular gene sequence which has a high level ofexpression. Alternatively, promoters from mammalian expression products,such as actin, collagen, myosin, and the like, may be employed.Transcriptional initiation regulatory signals may be selected whichallow for repression or activation, so that expression of the genesequences can be modulated. Of interest are regulatory signals which aretemperature-sensitive so that by varying the temperature, expression canbe repressed or initiated, or are subject to chemical (such asmetabolite) regulation.

Expression of PTP04 in eukaryotic hosts requires the use of eukaryoticregulatory regions. Such regions will, in general, include a promoterregion sufficient to direct the initiation of RNA synthesis. Preferredeukaryotic promoters include, for example, the promoter of the mousemetallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen.1:273-288, 1982); the TK promoter of Herpes virus (McKnight, Cell31:355-365, 1982); the SV40 early promoter (Benoist et al., Nature(London) 290:304-310, 1981); the yeast gal4 gene sequence promoter(Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975, 1982);Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955, 1984).

Translation of eukaryotic mRNA is initiated at the codon which encodesthe first methionine. For this reason, it is preferable to ensure thatthe linkage between a eukaryotic promoter and a DNA sequence whichencodes PTP04 (or a functional derivative thereof) does not contain anyintervening codons which are capable of encoding a methionine (i.e.,AUG). The presence of such codons results either in a formation of afusion protein (if the AUG codon is in the same reading frame as a PTP04coding sequence) or a frame-shift mutation (if the AUG codon is not inthe same reading frame as a PTP04 coding sequence).

A PTP04 nucleic acid molecule and an operably linked promoter may beintroduced into a recipient prokaryotic or eukaryotic cell either as anonreplicating DNA (or RNA) molecule, which may either be a linearmolecule or, more preferably, a closed covalent circular molecule (aplasmid). Since such molecules are incapable of autonomous replication,the expression of the gene may occur through the transient expression ofthe introduced sequence. Alternatively, permanent or stable expressionmay occur through the integration of the introduced DNA sequence intothe host chromosome.

A vector may be employed which is capable of integrating the desiredgene sequences into the host cell chromosome. Cells which have stablyintegrated the introduced DNA into their chromosomes can be selected byalso introducing one or more markers which allow for selection of hostcells which contain the expression vector. The marker may provide forprototrophy to an auxotrophic host, biocide resistance, e.g.,antibiotics, or heavy metals, such as copper, or the like. Theselectable marker gene sequence can either be directly linked to the DNAgene sequences to be expressed, or introduced into the same cell byco-transfection. Additional elements may also be needed for optimalsynthesis of single chain binding protein mRNA. These elements mayinclude splice signals, as well as transcription promoters, enhancers,and termination signals. cDNA expression vectors incorporating suchelements include those described by Okayama, Mol. Cell. Bio. 3:280,1983.

The introduced nucleic acid molecule can be incorporated into a plasmidor viral vector capable of autonomous replication in the recipient host.Any of a wide variety of vectors may be employed for this purpose.Factors of importance in selecting a particular plasmid or viral vectorinclude: the ease with which recipient cells that contain the vector maybe recognized and selected from those recipient cells which do notcontain the vector; the number of copies of the vector which are desiredin a particular host; and whether it is desirable to be able to“shuttle” the vector between host cells of different species.

Preferred prokaryotic vectors include plasmids such as those capable ofreplication in E. coil (such as, for example, pBR322, ColEl, pSC101,pACYC 184, pVX. Such plasmids are, for example, disclosed by Sambrook(cf. “Molecular Cloning: A Laboratory Manual”, second edition, edited bySambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, (1989)).Bacillus plasmids include pC194, pC221, pT127, and the like. Suchplasmids are disclosed by Gryczan (In: The Molecular Biology of theBacilli, Academic Press, NY (1982), pp. 307-329). Suitable Streptomycesplasmids include p1J101 (Kendall et al., J. Bacteriol.169:4177-4183,1987), and streptomyces bacteriophages such as fC31(Chater et al., In: Sixth International Symposium on ActinomycetalesBiology, Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54).Pseudomonas plasmids are reviewed by John et al. (Rev. Infect. Dis.8:693-704, 1986), and Izaki (Jpn. J. Bacteriol. 33:729-742, 1978).

Preferred eukaryotic plasmids include, for example, BPV, vaccinia, SV40,2-micron circle, and the like, or their derivatives. Such plasmids arewell known in the art (Botstein et al., Miami Wntr. Symp. 19:265-274,1982); Broach, In: “The Molecular Biology of the Yeast Saccharomyces:Life Cycle and Inheritance”, Cold Spring Harbor Laboratory, Cold SpringHarbor, NY, p. 445-470 (1981); Broach, Cell 28:203-204, 1982); Bollon etat., J. Clin. Hematol. Oncol. 10:39-48, 1980); Maniatis, In: CellBiology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression,Academic Press, NY, pp. 563-608 (1980).

Once the vector or nucleic acid molecule containing the construct(s) hasbeen prepared for expression, the DNA construct(s) may be introducedinto an appropriate host cell by any of a variety of suitable means,i.e., transformation, transfection, conjugation, protoplast fusion,electroporation, particle gun technology, calciumphosphate-precipitation, direct microinjection, and the like. After theintroduction of the vector, recipient cells are grown in a selectivemedium, which selects for the growth of vector-containing cells.Expression of the cloned gene molecule(s) results in the production ofPTP04 or fragments or functional derivatives thereof. This can takeplace in the transformed cells as such, or following the induction ofthese cells to differentiate (for example, by administration ofbromodeoxyuracil to neuroblastoma cells or the like). A variety ofincubation conditions can be used to form the peptide of the presentinvention. The most preferred conditions are those which mimicphysiological conditions.

V. PTP04 Polypeptides

Also a feature of the invention are PTP04 polypeptides. A variety ofmethodologies known in the art can be utilized to obtain thepolypeptides of the present invention. They may be purified from tissuesor cells which naturally produce them. Alternatively, theabove-described isolated nucleic acid sequences can be used to express aPTP04 protein recombinantly.

Any eukaryotic organism can be used as a source for the polypeptide ofthe invention, as long as the source organism naturally contains such apolypeptide. As used herein, “source organism” refers to the originalorganism from which the amino acid sequence is derived, regardless ofthe organism the protein is expressed in and ultimately isolated from.

One skilled in the art can readily follow known methods for isolatingproteins in order to obtain the peptide free of natural contaminants.These include, but are not limited to: size-exclusion chromatography,HPLC, ion-exchange chromatography, and immuno-affinity chromatography.

A PTP04 protein, like all proteins, is comprised of distinct functionalunits or domains. In eukaryotes, proteins sorted through the so-calledvesicular pathway (bulk flow) usually have a signal sequence (alsocalled a leader peptide) in the N-terminus, which is cleaved off afterthe translocation through the ER (endoplasmic reticulum) membrane. SomeN-terminal signal sequences are not cleaved off, remaining astransmembrane segments, but it does not mean these proteins are retainedin the ER; they can be further sorted and included in vesicles.Non-receptor proteins generally function to transmit signals within thecell, either by providing sites for protein:protein interactions or byhaving some catalytic activity (contained within a catalytic domain),often both. Methods of predicting the existence of these various domainsare well known in the art. Protein:protein interaction domains can beidentified by comparison to other proteins. The SH2 domain, for exampleis a protein domain of about 100 amino acids first identified as aconserved sequence region between the proteins Src and Fps (Sadowski, etal, Mol. Cell. Bio. 6:4396, 1986). Similar sequences were later found inmany other intracellular signal-transducing proteins. SH2 domainsfunction as regulatory modules of intracellular signaling cascades byinteracting with high affinity to phosphotyrosine-containing proteins ina sequence specific and strictly phosphorylation-dependent manner (Mayerand Baltimore, Trends Cell. Biol. 3:8, 1993). Kinase or phosphatasecatalytic domains can be identified by comparison to other knowncatalytic domains with kinase or phosphatase activity. See, for exampleHanks and Hunter, FASEB J. 9:576-595, 1995.

Primary sequence analysis of the PTP04 amino acid sequence (shown in SEQID NO: 2) reveals that it does not contain a signal sequence ortransmembrane domain and is, therefore, an intracellular protein.Comparison to known protein sequences revels that PTP04 is comprised ofseveral unique domains. These include a 48 amino acid N-terminal domain(shown from amino acid number 1-48 of SEQ ID NO:2), a 245 amino acidcatalytic domain (shown from amino acid number 49-294 of SEQ ID NO:2),and a 512 amino acid C-terminal domain (shown from amino acid number295-807 of SEQ ID NO:2).

These PTP04 domains have a variety of uses. An example of such a use isto make a polypeptide consisting of the PTP04 catalytic domain and aheterologous protein such as glutathione S-transferase (GST). Such apolypeptide can be used in a biochemical assay for PTP04 catalyticactivity useful for studying PTP04 substrate specificity or foridentifying substances that can modulate PTP04 catalytic activity.Alternatively, one skilled in the art could create a PTP04 polypeptidelacking at least one of the three major domains. Such a polypeptide,when expressed in a cell, is able to form complexes with the naturalbinding partner(s) of PTP04 but unable to transmit any signal furtherdownstream into the cell, i.e.,. it would be signaling incompetent andthus would be useful for studying the biological relevance of PTP04activity. (See, for example, Gishizky, et al, PNAS :10889, 1995).

VI. An Antibody Having Binding Affinity to a PTP04 Polypeptide and aHybridoma Containing the Antibody.

The present invention also relates to an antibody having specificbinding affinity to a PTP04 polypeptide. The polypeptide may have theamino acid sequence set forth in SEQ ID NO:2, or a be fragment thereof,or at least 6 contiguous amino acids thereof. Such an antibody may beidentified by comparing its binding affinity to a PTP04 polypeptide withits binding affinity to another polypeptide. Those which bindselectively to PTP04 would be chosen for use in methods requiring adistinction between PTP04 and other polypeptides. Such methods couldinclude, but should not be limited to, the analysis of altered PTP04expression in tissue containing other polypeptides and assay systemsusing whole cells.

A PTP04 peptide of the present invention can be used to produceantibodies or hybridomas. One skilled in the art will recognize that ifan antibody is desired, such a peptide would be generated as describedherein and used as an immunogen. Preferred PTP04 peptides for thispurpose as shown in Example 4 below. The antibodies of the presentinvention include monoclonal and polyclonal antibodies, as wellfragments of these antibodies, and humanized forms. Humanized forms ofthe antibodies of the present invention may be generated using one ofthe procedures known in the art such as chimerization or CDR grafting.The present invention also relates to a hybridoma which produces theabove-described monoclonal antibody, or binding fragment thereof. Ahybridoma is an immortalized cell line which is capable of secreting aspecific monoclonal antibody.

In general, techniques for preparing monoclonal antibodies andhybridomas are well known in the art (Campbell, “Monoclonal AntibodyTechnology: Laboratory Techniques in Biochemistry and MolecularBiology,” Elsevier Science Publishers, Amsterdam, The Netherlands, 1984;St. Groth et al., J. Immunol. Methods 35:1-21, 1980). Any animal (mouse,rabbit, and the like) which is known to produce antibodies can beimmunized with the selected polypeptide. Methods for immunization arewell known in the art. Such methods include subcutaneous orintraperitoneal injection of the polypeptide. One skilled in the artwill recognize that the amount of polypeptide used for immunization willvary based on the animal which is immunized, the antigenicity of thepolypeptide and the site of injection.

The polypeptide may be modified or administered in an adjuvant in orderto increase the peptide antigenicity. Methods of increasing theantigenicity of a polypeptide are well known in the art. Such proceduresinclude coupling the antigen with a heterologous protein (such asglobulin or b-galactosidase) or through the inclusion of an adjuvantduring immunization.

For monoclonal antibodies, spleen cells from the immunized animals areremoved, fused with myeloma cells, such as SP2/0-Agl4 myeloma cells, andallowed to become monoclonal antibody producing hybridoma cells. Any oneof a number of methods well known in the art can be used to identify thehybridoma cell which produces an antibody with the desiredcharacteristics. These include screening the hybridomas with an ELISAassay, western blot analysis, or radioimmunoassay (Lutz, et al., Exp.Cell Res. 175:109-124, 1988). Hybridomas secreting the desiredantibodies are cloned and the class and subclass is determined usingprocedures known in the art (Campbell, “Monoclonal Antibody Technology:Laboratory Techniques in Biochemistry and Molecular Biology”, supra,1984).

For polyclonal antibodies, antibody containing antisera is isolated fromthe immunized animal and is screened for the presence of antibodies withthe desired specificity using one of the above-described procedures. Theabove-described antibodies may be detectably labeled. Antibodies can bedetectably labeled through the use of radioisotopes, affinity labels(such as biotin, avidin, and the like), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, and the like) fluorescentlabels (such as FITC or rhodamine, and the like), paramagnetic atoms,and the like. Procedures for accomplishing such labeling are well-knownin the art, for example, see (Stemberger, et al., J. Histochem.Cytochem. 18:315, 1970; Bayer, et al., Meth. Enzym. 62:308, 1979;Engval, et al., Immunot. 109:129, 1972; Goding, J. Immunol. Meth.13:215, 1976). The labeled antibodies of the present invention can beused for in vitro, in vivo, and in in situ assays to identify cells ortissues which express a specific peptide.

The above-described antibodies may also be immobilized on a solidsupport. Examples of such solid supports include plastics such aspolycarbonate, complex carbohydrates such as agarose and sepharose,acrylic resins and such as polyacrylamide and latex beads. Techniquesfor coupling antibodies to such solid supports are well known in the art(Weir et al., “Handbook of Experimental Immunology” 4th Ed., BlackwellScientific Publications, Oxford, England, Chapter 10, 1986; Jacoby etal., Meth. Enzym. 34, Academic Press, N.Y., 1974). The immobilizedantibodies of the present invention can be used for in vitro, in vivo,and in situ assays as well as in immunochromotography.

Furthermore, one skilled in the art can readily adapt currentlyavailable procedures, as well as the techniques, methods and kitsdisclosed above with regard to antibodies, to generate peptides capableof binding to a specific peptide sequence in order to generaterationally designed antipeptide peptides, for example see Hurby et al.,“Application of Synthetic Peptides: Antisense Peptides”, In SyntheticPeptides, A User's Guide, W. H. Freeman, NY, pp. 289-307(1992), andKaspczak et al., Biochemistry 28:9230-8(1989).

VII. An Antibody Based Method and Kit for Detecting PTP04.

The present invention encompasses a method of detecting a PTP04polypeptide in a sample, comprising: (a) contacting the sample with anabove-described antibody, under conditions such that immunocomplexesform, and (b) detecting the presence of said antibody bound to thepolypeptide. In detail, the methods comprise incubating a test samplewith one or more of the antibodies of the present invention and assayingwhether the antibody binds to the test sample. Altered levels, either anincrease or decrease, of PTP04 in a sample as compared to normal levelsmay indicate disease.

Conditions for incubating an antibody with a test sample vary.Incubation conditions depend on the format employed in the assay, thedetection methods employed, and the type and nature of the antibody usedin the assay. One skilled in the art will recognize that any one of thecommonly available immunological assay formats (such asradioimmunoassays, enzyme-linked immunosorbent assays, diffusion basedOuchterlony, or rocket immunofluorescent assays) can readily be adaptedto employ the antibodies of the present invention. Examples of suchassays can be found in Chard, “An Introduction to Radioimmunoassay andRelated Techniques” Elsevier Science Publishers, Amsterdam, TheNetherlands (1986); Bullock et al., “Techniques in Immunocytochemistry,”Academic Press, Orlando, Fla. Vol. 1(1982), Vol. 2 (1983), Vol. 3(1985); Tijssen, “Practice and Theory of Enzyme Immunoassays: LaboratoryTechniques in Biochemistry and Molecular Biology,” Elsevier SciencePublishers, Amsterdam, The Netherlands (1985).

The immunological assay test samples of the present invention includecells, protein or membrane extracts of cells, or biological fluids suchas blood, serum, plasma, or urine. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing protein extracts or membraneextracts of cells are well known in the art and can be readily beadapted in order to obtain a sample which is capable with the systemutilized.

A kit contains all the necessary reagents to carry out the previouslydescribed methods of detection. The kit may comprise: (i) a firstcontainer containing an above-described antibody, and (ii) secondcontainer containing a conjugate comprising a binding partner of theantibody and a label. In another preferred embodiment, the kit furthercomprises one or more other containers comprising one or more of thefollowing: wash reagents and reagents capable of detecting the presenceof bound antibodies.

Examples of detection reagents include, but are not limited to, labeledsecondary antibodies, or in the alternative, if the primary antibody islabeled, the chromophoric, enzymatic, or antibody binding reagents whichare capable of reacting with the labeled antibody. The compartmentalizedkit may be as described above for nucleic acid probe kits. One skilledin the art will readily recognize that the antibodies described in thepresent invention can readily be incorporated into one of theestablished kit formats which are well known in the art.

VIII. Isolation of Natural Binding Partners of PTP04.

The present invention also relates to methods of detecting naturalbinding partners capable of binding to a PTP04 polypeptide. A naturalbinding partner of PTP04 may be, for example, a substrate protein whichis dephosphorylated as part of a signaling cascade. The bindingparter(s) may be present within a complex mixture, for example, serum,body fluids, or cell extracts.

In general methods for identifying natural binding partners compriseincubating a substance with PTP04 and detecting the presence of asubstance bound to PTP04. Preferred methods include the two-hybridsystem of Fields and Song (supra) and co-immunoprecipitation.

IX. Identification of and Uses for Substances Capable of ModulatingPTP04 Activity

The present invention also relates to a method of detecting a substancecapable of modulating PTP04 activity. Such substances can either enhanceactivity (agonists) or inhibit activity (antagonists). Agonists andantagonists can be peptides, antibodies, products from natural sourcessuch as fungal or plant extracts or small molecular weight organiccompounds. In general, small molecular weight organic compounds arepreferred. Examples of classes of compounds that can be tested for PTP04modulating activity are, for example but not limited to, thiazoles (seefor example co-pending U.S. application No. 60/033,522, Ser. No.08/660,900), and naphthopyrones (U.S. Pat. No. 5,602,171).

In general the method comprises incubating cells that produce PTP04 inthe presence of a test substance and detecting changes in the level ofPTP04 activity or PTP04 binding partner activity. A change in activitymay be manifested by increased or decreased phosphorylation of a PTP04polypeptide, increased or decreased phosphorylation of a PTP04substrate, or increased or decreased biological response in cells. Amethod for detecting modulation of PTP04 activity using thephosphorylation of an artificial substrate is shown in the examplesbelow. Biological responses can include, for example, proliferation,differentiation, survival, or motility. The substance thus identifiedwould produce a change in activity indicative of the agonist orantagonist nature of the substance. Once the substance is identified itcan be isolated using techniques well known in the art, if not alreadyavailable in a purified form.

The present invention also encompasses a method of agonizing(stimulating) or antagonizing PTP04 associated activity in a mammalcomprising administering to said mammal an agonist or antagonist toPTP04 in an amount sufficient to effect said agonism or antagonism. Alsoencompassed in the present application is a method of treating diseasesin a mammal with an agonist or antagonist of PTP04-related activitycomprising administering the agonist or antagonist to a mammal in anamount sufficient to agonize or antagonize PTP04 associated function(s).The particular compound can be administered to a patient either byitself or in a pharmaceutical composition where it is mixed withsuitable carriers or excipient(s). In treating a patient atherapeutically effective dose of the compound is administered. Atherapeutically effective dose referes to that amount of the compoundthat results in amelioration of symptoms or a prolongation of survivalin a patient.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals. For example, for determining the LD₅₀ (the dose lethal to 50%of the population) and the ED₅₀ (the dose therapeutically effective in50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Compounds which exhibit large therapeutic indices arepreferred. The data obtained from these cell culture assays and animalstudies can be used in formulating a range of dosage for use in human.The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating plasma concentration range that includes theIC₅₀ as determined in cell culture (i.e., the concentration of the testcompound which achieves a half-maximal disruption of the proteincomplex, or a half-maximal inhibition of the cellular level and/oractivity of a complex component). Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by HPLC.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (Seee.g. Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”,Ch. 1 p1).

It should be noted that the attending physician would know how to andwhen to terminate, interrupt, or adjust administration due to toxicity,or to organ dysfunctions. Conversely, the attending physician would alsoknow to adjust treatment to higher levels if the clinical response werenot adequate (precluding toxicity). The magnitude of an administrateddose in the management of the oncogenic disorder of interest will varywith the severity of the condition to be treated and to the route ofadministration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above may be used in veterinary medicine.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in “Remington'sPharmaceutical Sciences,” 1990, 18th ed., Mack Publishing Co., Easton,Pa. Suitable routes may include oral, rectal, transdermal, vaginal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections, just to name afew.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Forsuch transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

Use of pharmaceutically acceptable carriers to formulate the compoundsherein disclosed for the practice of the invention into dosages suitablefor systemic administration is within the scope of the invention. Withproper choice of carrier and suitable manufacturing practice, thecompositions of the present invention, in particular, those formulatedas solutions, may be administered parenterally, such as by intravenousinjection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated.

Agents intended to be administered intracellularly may be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents may be encapsulated into liposomes, thenadministered as described above. Liposomes are spherical lipid bilayerswith aqueous interiors. All molecules present in an aqueous solution atthe time of liposome formation are incorporated into the aqueousinterior. The liposomal contents are both protected from the externalmicroenvironment and, because liposomes fuse with cell membranes, areefficiently delivered into the cell cytoplasm. Additionally, due totheir hydrophobicity, small organic molecules may be directlyadministered intracellularly.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose,sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

X. Transgenic Animals.

Also contemplated by the invention are transgenic animals useful for thestudy of PTP04 activity in complex in vivo systems. A variety of methodsare available for the production of transgenic animals associated withthis invention. DNA sequences encoding PTP04 can be injected into thepronucleus of a fertilized egg before fusion of the male and femalepronuclei, or injected into the nucleus of an embryonic cell (e.g., thenucleus of a two-cell embryo) following the initiation of cell division(Brinster, et al., Proc. Nat. Acad. Sci. USA 82: 4438, 1985). Embryoscan be infected with viruses, especially retroviruses, modified to carryinorganic-ion receptor nucleotide sequences of the invention.

Pluripotent stem cells derived from the inner cell mass of the embryoand stabilized in culture can be manipulated in culture to incorporatenucleotide sequences of the invention. A transgenic animal can beproduced from such cells through implantation into a blastocyst that isimplanted into a foster mother and allowed to come to term. Animalssuitable for transgenic experiments can be obtained from standardcommercial sources such as Charles River (Wilmington, Mass.), Taconic(Germantown, N.Y.), Harlan Sprague Dawley (Indianapolis, Ind.), etc.

The procedures for manipulation of the rodent embryo and formicroinjection of DNA into the pronucleus of the zygote are well knownto those of ordinary skill in the art (Hogan, et al., supra).Microinjection procedures for fish, amphibian eggs and birds aredetailed in Houdebine and Chourrout, Experientia 47: 897-905, 1991).Other procedures for introduction of DNA into tissues of animals aredescribed in U.S. Pat. No. 4,945,050 (Sandford et al., Jul. 30, 1990).

By way of example only, to prepare a transgenic mouse, female mice areinduced to superovulate. After being allowed to mate, the females aresacrificed by CO, asphyxiation or cervical dislocation and embryos arerecovered from excised oviducts. Surrounding cumulus cells are removed.Pronuclear embryos are then washed and stored until the time ofinjection. Randomly cycling adult female mice are paired withvasectomized males. Recipient females are mated at the same time asdonor females. Embryos then are transferred surgically. The procedurefor generating transgenic rats is similar to that of mice. See Hammer,et al., Cell 63:1099-1112, 1990).

Methods for the culturing of embryonic stem (ES) cells and thesubsequent production of transgenic animals by the introduction of DNAinto ES cells using methods such as electroporation, calciumphosphate/DNA precipitation and direct injection also are well known tothose of ordinary skill in the art. See, for example, “Teratocarcinomasand Embryonic Stem Cells, A Practical Approach”, E. J. Robertson, ed.,IRL Press, 1987).

In cases involving random gene integration, a clone containing thesequence(s) of the invention is co-transfected with a gene encodingresistance. Alternatively, the gene encoding neomycin resistance isphysically linked to the sequence(s) of the invention. Transfection andisolation of desired clones are carried out by any one of severalmethods well known to those of ordinary skill in the art (E. J.Robertson, supra).

DNA molecules introduced into ES cells can also be integrated into thechromosome through the process of homologous recombination. Capecchi,Science 244: 1288-1292 (1989). Methods for positive selection of therecombination event (i.e., neo resistance) and dual positive-negativeselection (i.e., neo resistance and gancyclovir resistance) and thesubsequent identification of the desired clones by PCR have beendescribed by Capecchi, supra and Joyner et al., Nature 338: 153-156,1989), the teachings of which are incorporated herein. The final phaseof the procedure is to inject targeted ES cells into blastocysts and totransfer the blastocysts into pseudopregnant females. The resultingchimeric animals are bred and the offspring are analyzed by Southernblotting to identify individuals that carry the transgene. Proceduresfor the production of non-rodent mammals and other animals have beendiscussed by others. See Houdebine and Chourrout, supra; Pursel, et al.,Science 244:1281-1288, 1989); and Simms, et al., Bio/Technology6:179-183, 1988).

Thus, the invention provides transgenic, nonhuman mammals containing atransgene encoding a PTP04 polypeptide or a gene effecting theexpression of a PTP04 polypeptide. Such transgenic nonhuman mammals areparticularly useful as an in vivo test system for studying the effectsof introducing a PTP04 polypeptide, regulating the expression of a PTP04polypeptide (i.e., through the introduction of additional genes,antisense nucleic acids, or ribozymes).

A “transgenic animal” is an animal having cells that contain DNA whichhas been artificially inserted into a cell, which DNA becomes part ofthe genome of the animal which develops from that cell. Preferredtransgenic animals are primates, mice, rats, cows, pigs, horses, goats,sheep, dogs and cats. The transgenic DNA may encode for a human PTP04polypeptide. Native expression in an animal may be reduced by providingan amount of anti-sense RNA or DNA effective to reduce expression of thereceptor.

XI. Gene Therapy

PTP04 or its genetic sequences, both mutated and non-mutated, will alsobe useful in gene therapy (reviewed in Miller, Nature 357:455-460,(1992). Miller states that advances have resulted in practicalapproaches to human gene therapy that have demonstrated positive initialresults. The basic science of gene therapy is described in Mulligan,Science 260:926-931, (1993).

In one preferred embodiment, an expression vector containing a PTP04coding sequence or a PTP04 mutant coding sequence as described above isinserted into cells, the cells are grown in vitro and then infused inlarge numbers into patients. In another preferred embodiment, a DNAsegment containing a promoter of choice (for example a strong promoter)is transferred into cells containing an endogenous PTP04 in such amanner that the promoter segment enhances expression of the endogenousPTP04 gene (for example, the promoter segment is transferred to the cellsuch that it becomes directly linked to the endogenous PTP04 gene).

The gene therapy may involve the use of an adenovirus containing PTP04cDNA targeted to an appropriate cell type, systemic PTP04 increase byimplantation of engineered cells, injection with PTP04 virus, orinjection of naked PTP04 DNA into appropriate cells or tissues, forexample neurons.

Expression vectors derived from viruses such as retroviruses, vacciniavirus, adenovirus, adeno-associated virus, herpes viruses, several RNAviruses, or bovine papilloma virus, may be used for delivery ofnucleotide sequences (e.g., cDNA) encoding recombinant PTP04 proteininto the targeted cell population (e.g., tumor cells or neurons).Methods which are well known to those skilled in the art can be used toconstruct recombinant viral vectors containing coding sequences. See,for example, the techniques described in Maniatis et al., “MolecularCloning: A Laboratory Manual”, Cold Spring Harbor Laboratory, N.Y.(1989), and in Ausubel et al., “Current Protocols in Molecular Biology”,Greene Publishing Associates and Wiley Interscience, N.Y. (1989).Alternatively, recombinant nucleic acid molecules encoding proteinsequences can be used as naked DNA or in reconstituted system e.g.,liposomes or other lipid systems for delivery to target cells (See e.g.,Felgner et al., Nature 337:387-8, 1989). Several other methods for thedirect transfer of plasmid DNA into cells exist for use in human genetherapy and involve targeting the DNA to receptors on cells bycomplexing the plasmid DNA to proteins. See, Miller, supra.

In its simplest form, gene transfer can be performed by simply injectingminute amounts of DNA into the nucleus of a cell, through a process ofmicroinjection. (Capecchi M R, Cell 22:479-88, 1980). Once recombinantgenes are introduced into a cell, they can be recognized by the cellsnormal mechanisms for transcription and translation, and a gene productwill be expressed. Other methods have also been attempted forintroducing DNA into larger numbers of cells. These methods include:transfection, wherein DNA is precipitated with CaPO, and taken intocells by pinocytosis (Chen C. and Okayama H, Mol. Cell Biol. 7:2745-52,1987); electroporation, wherein cells are exposed to large voltagepulses to introduce holes into the membrane (Chu G., et al., NucleicAcids Res., 15:1311-26, 1987); lipofection/liposome fusion, wherein DNAis packaged into lipophilic vesicles which fuse with a target cell(Felgner P L., et al., Proc. Natl. Acad. Sci. USA. 84:7413-7, 1987));and particle bombardment using DNA bound to small projectiles (Yang N S.et al., Proc. Natl. Acad. Sci. 87:9568-72, 1990). Another method forintroducing DNA into cells is to couple the DNA to chemically modifiedproteins.

It has also been shown that adenovirus proteins are capable ofdestabilizing endosomes and enhancing the uptake of DNA into cells. Theadmixture of adenovirus to solutions containing DNA complexes, or thebinding of DNA to polylysine covalently attached to adenovirus usingprotein crosslinking agents substantially improves the uptake andexpression of the recombinant gene. Curiel D T et al., Am. J. Respir.Cell. Mol. Biol., 6:247-52, 1992).

As used herein “gene transfer” means the process of introducing aforeign nucleic acid molecule into a cell. Gene transfer is commonlyperformed to enable the expression of a particular product encoded bythe gene. The product may include a protein, polypeptide, anti-sense DNAor RNA, or enzymatically active RNA. Gene transfer can be performed incultured cells or by direct administration into animals. Generally genetransfer involves the process of nucleic acid contact with a target cellby non-specific or receptor mediated interactions, uptake of nucleicacid into the cell through the membrane or by endocytosis, and releaseof nucleic acid into the cytoplasm from the plasma membrane or endosome.Expression may require, in addition, movement of the nucleic acid intothe nucleus of the cell and binding to appropriate nuclear factors fortranscription.

As used herein “gene therapy” is a form of gene transfer and is includedwithin the definition of gene transfer as used herein and specificallyrefers to gene transfer to express a therapeutic product from a cell invivo or in vitro. Gene transfer can be performed ex vivo on cells whichare then transplanted into a patient, or can be performed by directadministration of the nucleic acid or nucleic acid-protein complex intothe patient.

In another preferred embodiment, a vector having nucleic acid sequencesencoding a PTP04 is provided in which the nucleic acid sequence isexpressed only in specific tissue. Methods of achieving tissue-specificgene expression as set forth in International Publication No. WO93/09236, filed Nov. 3, 1992 and published May 13, 1993.

In all of the preceding vectors set forth above, a further aspect of theinvention is that the nucleic acid sequence contained in the vector mayinclude additions, deletions or modifications to some or all of thesequence of the nucleic acid, as defined above.

In another preferred embodiment, a method of gene replacement is setforth. “Gene replacement” as used herein means supplying a nucleic acidsequence which is capable of being expressed in vivo in an animal andthereby providing or augmenting the function of an endogenous gene whichis missing or defective in the animal.

XII. Compounds that Modulate the Function of PTP04 Proteins

In an effort to discover novel treatments for diseases, biomedicalresearchers and chemists have designed, synthesized, and testedmolecules that inhibit the function of protein kinases. Some smallorganic molecules form a class of compounds that modulate the functionof protein kinases. Examples of molecules that have been reported toinhibit the function of protein kinases include, but are not limited to,bis monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO92/20642, published Nov. 26, 1992 by Maguire et al.), vinylene-azaindolederivatives (PCT WO 94/14808, published Jul. 7, 1994 by Ballinari etal.), 1-cyclopropyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992),styryl compounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridylcompounds (U.S. Pat. No. 5,302,606), certain quinazoline derivatives (EPApplication No. 0 566 266 A1), seleoindoles and selenides (PCT WO94/03427, published Feb. 17, 1994 by Denny et al.), tricyclicpolyhydroxylic compounds (PCT WO 92/21660, published Dec. 10, 1992 byDow), and benzylphosphonic acid compounds (PCT WO 91/15495, publishedOct. 17, 1991 by Dow et al). The compounds that can traverse cellmembranes and are resistant to acid hydrolysis are potentiallyadvantageous therapeutics as they can become highly bioavailable afterbeing administered orally to patients. However, many of these proteinkinase inhibitors only weakly inhibit the function of protein kinases.In addition, many inhibit a variety of protein kinases and willtherefore cause multiple side-effects as therapeutics for diseases.

Some indolinone compounds, however, form classes of acid resistant andmembrane permeable organic molecules. WO 96/22976, published Aug. 1,1996 by Ballinari et al. describes hydrosoluble indolinone compoundsthat harbor tetralin, naphthalene, quinoline, and indole substituentsfused to the oxindole ring. These bicyclic substituents are in turnsubstituted with polar moieties including hydroxylated alkyl, phosphate,and ether moieties. U.S. patent application Ser. No. 08/702,232, filedAug. 23, 1996, entitled “Indolinone Combinatorial Libraries and RelatedProducts and Methods for the Treatment of Disease” by Tang et al. (Lyon& Lyon Docket No. 221/187) and Ser. No. 08/485,323, filed Jun. 7, 1995,entitled “Benzylidene-Z-Indoline Compounds for the Treatment of Disease”by Tang et al. (Lyon & Lyon Docket No. 223/298) and International PatentPublication WO 96/22976, published Aug. 1, 1996 by Ballinari et al., allof which are incorporated herein by reference in their entirety,including any drawings, describe indolinone chemical libraries ofindolinone compounds harboring other bicyclic moieties as well asmonocyclic moieties fused to the oxindole ring. applications Ser. No.08/702,232, filed Aug. 23, 1996, entitled “Indolinone CombinatorialLibraries and Related Products and Methods for the Treatment of Disease”by Tang et al. (Lyon & Lyon Docket No. 221/187), Ser. No. 08/485,323,filed Jun. 7, 1995, entitled “Benzylidene-Z-Indoline Compounds for theTreatment of Disease” by Tang et al. (Lyon & Lyon Docket No. 223/298),and WO 96/22976, published Aug. 1, 1996 by Ballinari et al. teachmethods of indolinone synthesis, methods of testing the biologicalactivity of indolinone compounds in cells, and inhibition patterns ofindolinone derivatives.

Other examples of substances capable of modulating PTP04 activityinclude, but are not limited to, tyrphostins, quinazolines,quinoxolines, and quinolines.

The quinazolines, tyrphostins, quinolines, and quinoxolines referred toabove include well known compounds such as those described in theliterature. For example, representative publications describingquinazoline include Barker et al., EPO Publication No. 0 520 722 Al;Jones et al., U.S. Pat. No. 4,447,608; Kabbe et al., U.S. Pat. No.4,757,072; Kaul and Vougioukas, U.S. Pat. No. 5, 316,553; Kreighbaum andComer, U.S. Pat. No. 4,343,940; Pegg and Wardleworth, EPO PublicationNo. 0 562 734 A1; Barker et al., Proc. of Am. Assoc. for Cancer Research32:327 (1991); Bertino, J. R., Cancer Research 3:293-304 (1979);Bertino, J. R., Cancer Research 9(2 part 1):293-304 (1979); Curtin etal., Br. J. Cancer 53:361-368 (1986); Fernandes et al., Cancer Research43:1117-1123 (1983); Ferris et al. J. Org. Chem. 44(2):173-178; Fry etal., Science 265:1093-1095 (1994); Jackman et al., Cancer Research51:5579-5586 (1981); Jones et al. J. Med. Chem. 29(6):1114-1118; Lee andSkibo, Biochemistry 26(23):7355-7362 (1987); Lemus et al., J. Org. Chem.54:3511-3518 (1989); Ley and Seng, Synthesis 1975:415-522 (1975);Maxwell et al., Magnetic Resonance in Medicine 17:189-196 (1991); Miniet al., Cancer Research 45:325-330 (1985); Phillips and Castle, J.Heterocyclic Chem. 17(19):1489-1596 (1980), Reece et al., CancerResearch 47(11):2996-2999 (1977); Sculier et al., Cancer Immunol. andImmunother. 23:A65 (1986); Sikora et al., Cancer Letters 23:289-295(1984); Sikora et al., Analytical Biochem. 172:344-355 (1988); all ofwhich are incorporated herein by reference in their entirety, includingany drawings.

Quinoxaline is described in Kaul and Vougioukas, U.S. Pat. No.5,316,553, incorporated herein by reference in its entirety, includingany drawings.

Quinolines are described in Dolle et al., J. Med. Chem. 37:2627-2629(1994); MaGuire, J. Med. Chem. 37:2129-2131 (1994); Burke et al., J.Med. Chem. 36:425-432 (1993); and Burke et al. BioOrganic Med. Chem.Letters 2:1771-1774 (1992), all of which are incorporated by referencein their entirety, including any drawings.

Tyrphostins are described in Allen et al., Clin. Exp. Immunol.91:141-156 (1993); Anafi et al., Blood 82:12:3524-3529 (1993); Baker etal., J. Cell Sci. 102:543-555 (1992); Bilder et al., Amer. Physiol. Soc.pp. 6363-6143:C721-C730 (1991); Brunton et al., Proceedings of Amer.Assoc. Cancer Rsch. 33:558 (1992); Bryckaert et al., Experimental CellResearch 199:255-261 (1992); Dong et al., J. Leukocyte Biology 53:53-60(1993); Dong et al., J. Immunol. 151(5):2717-2724 (1993); Gazit et al.,J. Med. Chem. 32:2344-2352 (1989); Gazit et al., J. Med. Chem.36:3556-3564 (1993); Kaur et al., Anti-Cancer Drugs 5:213-222 (1994);Kaur et al., King et al., Biochem. J. 275:413-418 (1991); Kuo et al.,Cancer Letters 74:197-202 (1993); Levitzki, A., The FASEB J. 6:3275-3282(1992); Lyall et al., J. Biol. Chem. 264:14503-14509 (1989); Peterson etal., The Prostate 22:335-345 (1993); Pillemer et al., Int. J. Cancer50:80-85 (1992); Posner et al., Molecular Pharmacology 45:673-683(1993); Rendu et al., Biol. Pharmacology 44(5):881-888 (1992); Sauro andThomas, Life Sciences 53:371-376 (1993); Sauro and Thomas, J. Pharm. andExperimental Therapeutics 267(3):119-1125 (1993); Wolbring et al., J.Biol. Chem. 269(36):22470-22472 (1994); and Yoneda et al., CancerResearch 51:4430-4435 (1991); all of which are incorporated herein byreference in their entirety, including any drawings.

Other compounds that could be used as modulators include oxindolinonessuch as those described in U.S. patent application Ser. No. 08/702,232filed Aug. 23, 1996, incorporated herein by reference in its entirety,including any drawings.

EXAMPLES Example 1

Isolation of cDNA Clones Encoding PTP04

The example below describes the isolation and identification of a newPTP sequence from primary cancer tissues and the subsequent cloning of afull-length human PTP04. Also described are probes useful for thedetection of PTP04 in cells or tissues.

Materials and Methods:

Poly A+ RNA was isolated from 30 uM cryostat sections of frozen samplesfrom primary human lung and colon carcinomas (Micro-FastTrack,InVitrogen, San Diego, Calif.). This RNA was used to generatesingle-stranded cDNA using the Superscript Preamplification System(GIBCO BRL, Gaithersburg, Md.; Gerard, G F et al. (1989), FOCUS 11, 66)under conditions recommended by the manufacturer. A typical reactionused 10 μg total RNA or 2 μg poly(A) RNA with 1.5 μg oligo(dT)₁₂₋₁₈ in areaction volume of 60 μL. The product was treated with RNaseH anddiluted to 100 μL with H₂O. For subsequent PCR amplification, 1-4 μL ofthis sscDNA was used in each reaction.

Degenerate oligonucleotides were synthesized on an Applied Biosystems394 DNA synthesizer using established phosphoramidite chemistry,precipitated with ethanol and used unpurified for PCR. The sequence ofthe degenerate oligonucleotide primers follows:

PTPDFW = 5′-GAYTTYTGGVRNATGRTNTGGGA- (sense) (SEQ ID NO:3) and PTPHCSA= 5′-CGGCCSAYNCCNGCNSWRCARTG -3′ (antisense) (SEQ ID NO:4).

These primers were derived from the peptide sequences DFWXMXW(E/D) (SEQID NO:5) (sense strand from PTP catalytic domain) and HCXAGXG (antisensestrand from PTP catalytic domain) (SEQ ID NO:6), respectively.Degenerate nucleotide residue designations are: N=A, C, G, or T; R=A orG; and Y=C or T.

PCR reactions were performed using degenerate primers applied to thesingle-stranded cDNA listed above. The primers were added at a finalconcentration of 5 μM each to a mixture containing 10 mM Tris.HCl(pH8.3), 50 mM KCl, 1.5 mM MgCl₂, 200 μM each deoxynucleosidetriphosphate, 0.001% gelatin, 1.5 U AmpliTaq DNA Polymerase(Perkin-Elmer/Cetus), and 1-4 μL cDNA. Following 3 min denaturation at95° C., the cycling conditions were 94° C. for 30 s, 50° C. for 1 min,and 72° C. for 1 min 45 s for 35 cycles. PCR fragments migrating between350-400 bp were isolated from 2% agarose gels using the GeneClean Kit(Bio101), and T-A cloned into the pCRII vector (Invitrogen Corp. U.S.A.)according to the manufacturer's protocol.

Colonies were selected for mini plasmid DNA-preparations using Qiagencolumns and the plasmid DNA was sequenced using cycle sequencingdye-terminator kit with AmpliTaq DNA Polymerase, FS (ABI, Foster City,Calif.). Sequencing reaction products were run on an ABI Prism 377 DNASequencer, and analyzed using the BLAST alignment algorithm (Altschul,S. F. et al., J. Mol. Biol. 215:403-10). One novel clone novel clone(G77-4a-117), designated PTP04, was isolated from human HLT370 primarylung carcinoma sample.

To obtain full-length cDNA encoding the novel phosphatase, RACE (rapidamplification of cDNA ends) was performed with sense or anti-senseoligonucleoides derived from the original PCR fragments. Marathon-ReadycDNA (Clontech, Palo Alto, Calif.) made from human Molt-4 leukemia cellswas used in the RACE reactions with the following

primers: RACE primers: 5′-CACCGTTCGAGTATTTCAGATTGTGAAGAAG-TCC-3′ (6595)(SEQ ID NO:7), 5′-GGACTTCTTCACAATCTGAAATACTCGAACGGTG-3′ (6596) (SEQ IDNO:8), 5′-CCGTTATGTGAGGAAGAGCCACATTACAGGACC-3′ (6599) (SEQ ID NO:9),5′-GGTCCTGTAATGTGGCTCTTCCTCACATAACGG-3′ (6600) (SEQ ID NO:10), AP-1, andAP-2 (Clontech). RT-PCR primers for PTP04: 5′-GGCATGCATGGAGTATGAAATGG-3′(6618) (SEQ ID NO:11), 5′-CGTACATCCCAGATGAGCTCAAGAATAGGG-3′ (6632) (SEQID NO:12).

Isolated cDNA fragments encoding PTP04 were confirmed by DNA sequencingand subsequently used as probes for the screening of a human leukocytecDNA library.

A human leukocyte cDNA library (lTriplEx, Clontech) and a Molt-4leukemia cell library (lgt11, Clontech) were then screened to isolatefull-length transcripts encoding PTP04. The 5′ or 3′-RACE fragments were³²P-labeled by random priming and used as hybridization probes at 2×10⁶cpm/mL following standard techniques for library screening.Pre-hybridization (3 h) and hybridization (overnight) were conducted at42° C. in 5×SSC, 5× Denhart's solution, 2.5% dextran sulfate, 50 mMNa₂PO₄/NaHPO₄ [pH 7.0], 50% formamide with 100 mg/mL denatured salmonsperm DNA. Stringent washes were performed at 65° C. in 0.1×SSC and 0.1%SDS. Several overlapping clones were isolated and found to span thesequence of the PCR fragment (G77-4a-117). The final sequence wasverified by sequencing of both strains using a cycle sequencingdye-terminator kit with AmpliTaq DNA Polymerase, FS (ABI, Foster City,Calif.). Sequencing reaction products were run on an ABI Prism 377 DNASequencer.

Results

The 3,580 bp human PTP04 nucleotide sequence encodes a polypeptide of807 amino acids. The PTP04 coding sequence is flanked by a 52 nucleotide5′-untranslated region and a 1086 nucleotide 3′-untranslated regionending with a poly(A) tail. While there are no upstream in frame stopcodons, the first ATG beginning at nucleotide position 53 conforms tothe Kozak consensus for an initiating methionine. This predicted first 6amino acids are identical to those of murine ZPEP (SwissProt: P29352,GeneBank: M90388), further supporting this is the true translationalstart site. One cDNA clone had an insert after nucleotide 30 in the5′UTR, but otherwise had no sequence differences.

The 807 amino acid sequence shows no signal sequence or a transmembranedomain and PTP04 is, therefore, an intracellular protein. PTP04 has anN-terminal region from amino acids 1-48, a catalytic domain from aminoacids 49-294, and a C-terminal tail from amino acids 295-807. PTP04 ismost related to murine ZPEP with an overall homology of 70%. ZPEP is amember of a subfamily of PTPs that includes PTP-PEST, HSC, BDP1 andPTP20, all of which are cytoplasmic PTPs with a single catalytic domainand a region rich in Pro, Glu, Ser and Thr residues (PEST domain). PTP04also has a C-terminal PEST domain, from amino acids 495-807, where thereare 57 serine residues (18%) and 35 proline residues(11%). A comparisonof the amino acid sequences of PTP04 and ZPEP is shown in FIG. 1.

The homology between PTP04 and ZPEP is concentrated in the N terminaland C-terminal ends of the proteins with significant divergence in themiddle. The N-terminal region of PTP04, from amino acids 1-48, is 81%homologous to murine ZPEP. The catalytic domain of PTP04, from aminoacids 49-294, is 89% homologous to murine ZPEP. The region of PTP04 from294-600 is approximately 50% homologous to murine ZPEP. The C-terminalregion of PTP04, from 680-817, is 80% homologous to murine ZPEP. Thehuman SuPTP04 sequence defines a novel member of the PTP-PEST subfamilyof PTPs.

Example 2

Expression of PTP04

The example below shows the evaluation of PTP04 expression in normalhuman tissues and in cancer cell lines.

Materials and Methods

Northern blots were prepared by running 20 μg total RNA per laneisolated from 22 human adult normal tissues (thymus, lung, duodenum,colon, testis, brain, cerebellum, salivary gland, heart, liver,pancreas, kidney, spleen, stomach, uterus, prostate, skeletal muscle,placenta, mammary gland, bladder, lymph node, adipose tissue), 2 humanfetal normal tissues (fetal liver, fetal brain), and 24 human tumor celllines (HOP-92, EKVX, NCI-H23, NCI-H226, NCI-H322M, NCI-H460, A549,HOP-62, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, IGROV1, SK-OV-3, SNB-19,SNB-75, U251, SF-268, SF-295, SF-539, CCRF-CEM, SR, DU-145, PC-3)(obtained from Nick Scuidero, National Cancer Institute, DevelopmentalTherapeutics Program, Rockville, Md.). The total RNA samples were run ona denaturing formaldehyde 1% agarose gel and transferred onto anitrocellulose membrane (BioRad, CA). An additional human normal tissueNorthern blot containing 2 μg polyA+mRNA per lane from 8 different humancancer cell lines (NCI-H522, K-562, MOLT-4, HL-60, S3, Raji, SW480,G361) on a charge-modified nylon membrane (human cancer cell line blot#7757-1, Clontech, Palo Alto, Calif.) were also hybridized.

For the total RNA samples, nitrocellulose membranes were hybridized withrandomly primed [a-³²P]dCTP-labeled probes synthesized from a 579 bpStuI-BstXI fragment of pCR2.1.mini298. Hybridization was performedovernight at 42° C. in 4×SSPE, 2.5× Denhardt's solution, 50% formamide,0.2 mg/mL denatured salmon sperm DNA, 0.1 mg/mL yeast tRNA (BoehringerMannheim,IN), 0.2% SDS, with 5×10⁶ cpm/mL of [a-³²P]dCTP labeled DNAprobes on a Techne hybridizer HB-1. The blots were washed with 2×SSC,0.1% SDS, at 65° C. for 20 min twice followed by in 0.5×SSC, 0.1% SDS at65° C. for 20 min. The blots were exposed to a phospho-imaging screenfor 24 hours and scanned on a Molecular Dynamics Phosphoimager SF.

A 351 bp EcoRI-HindIII fragment of G77-4a-117 was used to generate aprobe for 2 μg poly A+ mRNA samples on a Clontech nylon membrane.Hybridization was performed at 42° C. overnight in 5×SSC, 2% SDS, 10×Denhardt's solution, 50% formamide, 100 μg/mL denatured salmon sperm DNAwith 1-2×10⁶ cpm/mL of [a-³²P]dCTP -labeled DNA probes. The membrane waswashed at room temperature in 2× SSC/0.05% SDS for 30 min and followedby at 50° C. in 0.2× SSC/0.1% SDS for 30 min, twice, and exposed for 48hours on Kodak XAR-2 film.

RT-PCR Detection of Novel PTPs

Total RNA was isolated from various cell lines or fresh frozen tissuesby centrifugation thrugh a cesium chloride cushion. Twenty μg of totalRNA was reverse transcribed with random hexamers and Moloney murineleukemia virus reverse transcriptase (Super-ScriptII, GIBCO BRL,Gaithersburg, Md.). PCR was then used to amplify cDNA encoding SuPTP04.RT-PCR reactions lacking only the reverse transcriptase were performedas controls. PCR products were electrophoresed on 3% agarose gels,visualized by ethidium bromide staining and photographed on a UV lightbox. The intensity for a 270-bp fragment specific to PTP04 were comparedamong different RNA samples.

Results

A single SuPTP04 mRNA transcript of approximately 4.5 kb was identifiedby Northern analysis, and found to be exclusively in the Thymus. Therest of 23 human normal tissues (fetal brain, fetal liver, lung,duodenum, colon, testis, brain, cerebellum, salivary gland, heart,liver, pancreas, kidney, spleen, stomach, uterus, prostate, skeletalmuscle, placenta, mammary gland, bladder, lymph node, adipose tissue)were all negative. Six of the human tumor cell lines (CCRF-CEM, K-562,MOLT-4, HL-60, SR, Raji) were positive. The rest of 26 human tumor celllines were negative. RT-PCR with gene specific primer-pairs showed thatexpression of the transcripts encoding SuPTP04 confirmed the resultsfrom Northern analysis and also detected low levels in adipose, kidney,small intestine, hematopoietic tissues and various cell types (spleen,thymus, lymph node, bone marrow, peripheral leukocytes and lymphocytes.

The selective expression of PTP04 in cells of hematopoetic originincluding normal human thymus and several leukemia cell lines suggests apotential involvement in immune regulation including T and B cellsurvival, differentiation or co-stimulation, and/or inflammatory,immunosuppressive or autoimmune disorders. Additionally, expression inadipose tissue suggests a possible role in metabolic disorders such asdiabetes.

Example 3

Recombinant Expression of PTP04

The following example illustrates the construction of vectors forexpression of recombinant PTP04 and the creation of recombinant celllines expressing PTP04.

Construction of Expression Vectors

Expression constructs were generated by PCR-assisted mutagenesis inwhich the entire coding domains of PTP04 was tagged on itscarboxy-terminal end with the hemophilus influenza hemaglutinin (HA)epitope YPYDVPDYAS (SEQ ID NO:13) (Pati, 1992). The construct wasintroduced into two mammalian expression vectors: pLXSN (Miller, A. D. &Rosman, G. J., Biotechniques 7, 980-988, 1989) for the generation ofvirus producing lines; and pRKS for transient expression in mammalian.

Dominant negative (signaling incompetent) PTP04 constructs were alsomade in both pLXSN and PRK5 by mutation of the invariant Cys in theconserved HCSAG (SEQ ID NO:14) motif to an Ala by PCR mutagenesis.

The entire PTP04 open reading frames (no HA-tag) excluding theinitiating methionines were generated by PCR and ligated into pGEXvector (Pharmacia Biotech, Uppsala, Sweden) for bacterial production ofGST-fusion proteins for immunization of rabbits for antibody production.The entire PTP04 open reading frame excluding the initiating methionineswas generated by PCR and ligated into pGEX vector for bacterialproduction of GST-fusion proteins for immunization of rabbits forantibody production. This vector contains the glutathione-S-transferasecoding sequence followed by a polylinker for generating recombinantfusion proteins. The GST moiety comprises the N-terminal portion of thefusion protein.

Transient Expression in Mammalian Cells

The pRK5 expression plasmids (10 μg DNA/100 mm plate) containing theHA-tagged PTP04 gene can be introduced into COS and 293 cells withlipofectamine (Gibco BRL). After 72 hours, the cells were harvested in0.5 mL solubilization buffer (20 mM HEPES pH7.35, 150 mM NaCl, 10%glycerol, 1% Triton X-100, 1.5 mM MgCl,, 1 mM EGTA, 2 mMphenylmethylsulfonyl fluoride, 1 μg/mL aprotinin). Sample aliquots wereresolved by SDS polyacrylamide gel electrophoresis (PAGE) on 15%acrylamide/0.5% bis-acrylamide gels and electrophoretically transferredto nitrocellulose. Non-specific binding was blocked by preincubatingblots in Blotto (phosphate buffered saline containing 5% w/v non-fatdried milk and 0.2% v/v nonidet P-40 (Sigma)), and recombinant proteinwas detected using a murine Mab to the HA decapeptide tag.Alternatively, recombinant protein can be detected using variousPTP04-specific antisera.

Generation of Virus Producing Cell Lines

pLXSN recombinant constructs containing the PTP04 gene were transfectedinto an amphotropic helper cell line PA317 using CaCl₂ mediatedtransfection. After selection on G418, the cells were plated on normalmedia without G418 (500 μg/mL). Supernatants from resistant cells wereused to infect the ecotropic helper cell line GP+E86, and cells againselected on G418. Resistant cells were again taken off G418, and thesupernatants harvested every 8-12 hours and pooled as virus stock.Redemann et al., 1992, Mol. Cell. Biol. 12: 491-498. Viral stock titerswere typically ˜10⁶/mL.

Stable Expression in Mammalian Cells

NIH-3T3, and BALB/3T3 cells were grown in 100 mm plates with DMEM(Gibco) containing 10% fetal calf serum (FCS). The cells weresuperinfected with the PTP04 retrovirus by adding approximately 3 mLviral supernatant to 15 mL culture media for approximately 24 hours.Cells expressing the retroviral constructs were then selected by growthin DMEM/10% FCS supplemented with 500 μg/mL G418.

Example 4

Generation of Anti-PTP04 Antibodies

PTP04-specific immunoreagents were raised in rabbits against a mixtureof three KLH-conjugated synthetic peptides corresponding to uniquesequences present in human PTP04. The peptides (see below) wereconjugated at the C-terminal residue with KLH.

peptide 428A: SWPPSGTSSKMSLDDLPEKQDGTVFPSSLLP (SEQ ID NO:15) peptide429A: YSLPYDSKHQIRNASNVKHHDSSALGVYSY (SEQ ID NO:16) peptide 430A:HTLQADSYSPNLPKSTTKAAKMMNQQRTKC (SEQ ID NO:17)

Additional immunoreagents were generated by immunizing rabbits with thebacterially expressed entire coding region of PTP04 expressed as aGST-fusion protein. GST fusion proteins were produced in DH5-alpha E.coli bacteria as descaribed in Smith, et al. Gene 67:31, 1988. Bacterialprotein lysates were purified on glutathione-sepharose matrix asdescribed in Smith, et al., supra.

Example 5

Assay for PTP04 Activity

Materials and Methods:

Recombinant wild-type and dominant negative (signaling incompetent)PTP04 (see Example 3, supra) were purified from bacteria as GST-fusionproteins. Lysates were bound to a glutathione-sepharaose matrix andwashed twice with 1×HNTG, followed by one wash with a buffer containing100 mM 2-(N-morpholino)ethansulfonic acid (MES), pH 6.8, 150 mM NaCl,and 1 mM EDTA.

The assay for phosphatase activity was essentially done as described byPei et al.(1993) using p-nitrophenolphosphate (PNPP) as a generic PTPsubstrate. Briefly, after the last washing step, reactions were startedby adding 50 mL Assay Buffer (100 mM MES pH 6.8, 150 mM NaCl, 10 mM DTT,2 mM EDTA, and 50 mM PNPP) to the matrix bound proteins. Samples wereincubated for 20 min. at 23° C. The reactions were terminated by mixing40 μL of each sample with 960 μL 1 N NaOH, and the absorbance ofp-nitrophenol was determined at 450 nm. To control for the presence ofPTP04 in the precipitates, the precipitates were boiled in SDS samplebuffer and analyzed by SDS-PAGE. The presence of PTP04 was then detectedby immunoblot analysis with anti-PTP04 antibodies.

Example 6

Screening Systems for the Identification of Inhibitors of PTP04 Activity

Assays may be performed in vitro or in vivo and are described in detailherein or can be obtained by modifying existing assays, such as thegrowth assay described in patent application Ser. No. 08/487,088 (Lyon &Lyon Docket No. 212/276), filed Jun. 7, 1995, by Tang et al., andentitled “Novel Pharmaceutical Compounds,” or the assays described inpatent application Ser. No. 60/005,167 (Lyon & Lyon Docket No. 215/256),filed Oct. 13, 1995 by Seedorf et al., and entitled “Diagnosis andTreatment of TKA-1 related disorders,” all of which are herebyincorporated herein by reference in their entirety including anydrawings. Another assay which could be modified to use the genes of thepresent invention is described in International Application No. WO94/23039, published Oct. 13, 1994, hereby incorporated herein byreference in its entirety including any drawings. Other possibilitiesinclude detecting kinase activity in an autophosphorylation assay ortesting for kinase activity on standard substrates such as histones,myelin basic protein, gamma tubulin, or centrosomal proteins. Bindingpartners may be identified by putting the N-terminal portion of theprotein into a two-hybrid screen or detecting phosphotyrosine of a dualspecificity kinase (Fields and Song, U.S. Pat. No. 5,283,173, issuedFeb. 1, 1994, incorporated by reference herein, including any drawings).

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Themolecular complexes and the methods, procedures, treatments, molecules,specific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. It will be readily apparentto one skilled in the art that varying substitutions and modificationsmay be made to the invention disclosed herein without departing from thescope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising,” “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group. For example, if X isdescribed as selected from the group consisting of bromine, chlorine,and iodine, claims for X being bromine and claims for X being bromineand chlorine are fully described.

In view of the degeneracy of the genetic code, other combinations ofnucleic acids also encode the claimed peptides and proteins of theinvention. For example, all four nucleic acid sequences GCT, GCC, GCA,and GCG encode the amino acide alanine. Therefore, if for an amino acidthere exists an average of three codons, a polypeptide of 100 aminoacids in length will, on average, be encoded by 3¹⁰⁰, or 5×10⁴⁷ nucleicacid sequences. Thus, a nucleic acid sequence can be modified to form asecond nucleic acid sequence, encoding the same polypeptide as endodedby the first nucleic acid sequences, using routine procedures andwithout undue experimentation. Thus, all possible nucleic acids thatencode the claimed peptides and proteins are also fully describedherein, as if all were written out in full taking into account the codonusage, especially that preferred in humans. Furthermore, changes in theamino acid sequences of polypeptides, or in the corresponding nucleicacid sequence encoding such polypeptide, may be designed or selected totake place in an area of the sequence where the significant activity ofthe polypeptide remains unchanged. For example, an amino acid change maytake place within a β-turn, away from the active site of thepolypeptide. Also changes such as deletions (e.g. removal of a segmentof the polypeptide, or in the corresponding nucleic acid sequenceencoding such polypeptide, which does not affect the active site) andadditions (e.g. addition of more amino acids to the polypeptide sequencewithout affecting the function of the active site, such as the formationof GST-fusion proteins, or additions in the corresponding nucleic acidsequence encoding such polypeptide without affecting the function of theactive site) are also within the scope of the present invention. Suchchanges to the polypeptides can be performed by those with ordinaryskill in the art using routine procedures and without undueexperimentation. Thus, all possible nucleic and/or amino acid sequencesthat can readily be determined not to affect a significant activity ofthe peptide or protein of the invention are also fully described herein.

Other embodiments are within the following claims.

1. An isolated, enriched, or purified PTP04 polypeptide, wherein saidpolypeptide comprises: (a) an amino acid sequence comprising at least90% identity to the full length amino acid sequence set forth in SEQ IDNO: 2; (b) an amino acid sequence comprising a sequence having at least90% identity over the length of amino acid residues 49-294 of SEQ ID NO:2, and lacking amino acid residues 1-48 of SEQ ID NO: 2; (c) an aminoacid sequence comprising a sequence having at least 90% identity overthe length of amino acid residues 49-294 of SEQ ID NO:2; or (d) an aminoacid sequence comprising a sequence having at least 90% identity overthe length of amino acid residues 49-294 of SEQ ID NO:2 and lackingamino acid residues 1-48 or amino acid residues 295-807, wherein saidpolypeptide possesses an enzymatic activity of a tyrosine phosophatase.2. The polypeptide of claim 1, wherein said polypeptide comprises anamino acid sequence comprising at least 95% identity to the full lengthamino acid sequence set forth in SEQ ID NO:
 2. 3. The polypeptide ofclaim 1 wherein said polypeptide comprises the full length amino acidsequence set forth in SEQ ID NO:
 2. 4. The polypeptide of claim 1,wherein said polypeptide comprises a sequence having at least 95%identity over the length of amino acid residues 49-294 of SEQ ID NO: 2.5. The polypeptide of claim 1, wherein said polypeptide comprises theamino acid sequence set forth in amino acid residues 49-294 of SEQ IDNO:
 2. 6. The polypeptide of claim 1 wherein said polypeptide comprisesa sequence having at least 95% identity over the length of amino acidresidues 49-294 of SEQ ID NO:2 and lacking amino acid residues 1-48 or295-807 of SEQ ID NO:
 2. 7. The polypeptide of claim 1 wherein saidpolypeptide comprises the full length amino acid sequence set forth inSEQ ID NO: 2 except it lacks amino acid residues 1-48 or 295-407 of SEQID NO:2.
 8. An isolated, enriched or purified polypeptide comprising thepolypeptide of claim 1 fused to a second polypeptide.
 9. The isolated,enriched or purified polypeptide of claim 8, wherein said secondpolypeptide is hemagglutinin or GST.
 10. A composition comprising thepolypeptide of claim 1 and a pharmaceutically acceptable carrier.